Circadian disturbances by altering the light-dark cycle negatively affects hematopoietic function of bone marrow in mice.

Circadian rhythms in metabolically active tissues are crucial for maintaining physical health. Circadian disturbance (CD) can cause various health issues, such as metabolic abnormalities and immune and cognitive dysfunctions. However, studies on the role of CD in immune cell development and differentiation, as well as the rhythmic expression of the core clock genes and their altered expression under CD, remain unclear. Therefore, we exposed C57bl/6j mice to repeated reversed light-dark cycles for 90 days to research the effects of CD on bone marrow (BM) hematopoietic function. We also researched the effects of CD on endogenous circadian rhythms, temporally dependent expression in peripheral blood and myeloid leukocytes, environmental homeostasis within BM, and circadian oscillations of hematopoietic-extrinsic cues. Our results confirmed that when the light and dark cycles around mice were frequently reversed, the circadian rhythmic expression of the two main circadian rhythm markers, the hypothalamic clock gene, and serum melatonin, was disturbed, indicating that the body was in a state of endogenous CD. Furthermore, CD altered the temporally dependent expression of peripheral blood and BM leukocytes and destroyed environmental homeostasis within the BM as well as circadian oscillations of hematopoietic-extrinsic cues, which may negatively affect BM hematopoiesis in mice. Collectively, these results demonstrate that circadian rhythms are vital for maintaining health and suggest that the association between CD and hematopoietic dysfunction warrants further investigation.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities.

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

Impact of circadian clock protein Bmal1 on experimentally-induced periodontitis-associated renal injury.

OBJECTIVES: To investigate the mechanism of circadian clock protein Bmal1 (Bmal1) on renal injury with chronic periodontitis, we established an experimental rat periodontitis model. METHODS: Twelve male Wistar rats were randomly divided into control and periodontitis groups (n=6, each group). The first maxillary molars on both sides of the upper jaw of rats with periodontitis were ligated by using orthodontic ligature wires, whereas the control group received no intervention measures. After 8 weeks, clinical periodontal parameters, including probing depth, bleeding index, and tooth mobility, were evaluated in both groups. Micro-CT scanning and three-dimensional image reconstruction were performed on the maxillary bones of the rats for the assessment of alveolar bone resorption. Histopatholo-gical observations of periodontal and renal tissues were conducted using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Renal function indicators, such as creatinine, albumin, and blood urea nitrogen levels, and oxidative stress markers, including superoxide dismutase, glutathione, and malondialdehyde levels, were measured using biochemical assay kits. MitoSOX red staining was used to detect reactive oxygen species (ROS) content in the kidneys. The gene and protein expression levels of Bmal1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in rat renal tissues were assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. RESULTS: Micro-CT and HE staining results showed significant bone resorption and attachment loss in the maxillary first molar region of the periodontitis group. Histological examination through HE and PAS staining revealed substantial histopathological damage to the renal tissues of the rats in the periodontitis group. The findings of the assessment of renal function and oxidative stress markers indicated that the periodontitis group exhibited abnormal levels of oxidative stress, whereas the renal function levels showed abnormalities without statistical significance. MitoSOX Red staining results showed that the content of ROS in the renal tissue of the periodontitis group was significantly higher than that of the control group, and RT-qPCR and immunohistochemistry results showed that the expression levels of Bmal1, Nrf2, and HO-1 in the renal tissues of the rats in the periodontitis group showed a decreasing trend. CONCLUSIONS: Circadian clock protein Bmal1 plays an important role in the oxidative damage process involved in the renal of rats with periodontitis.

A Luciferase Imaging-Based Assay for Studying Temperature Compensation of the Circadian Clock.

The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.

Dissection of Daytime and Nighttime Thermoresponsive Hypocotyl Elongation in Arabidopsis.

Hypocotyl elongation in Arabidopsis is widely utilized as a readout for phytochrome B (phyB) signaling and thermomorphogenesis. Hypocotyl elongation is gated by the circadian clock and, therefore, it occurs at distinct times depending on day length or seasonal cues. In short-day conditions, hypocotyl elongation occurs mainly at the end of nighttime when phyB reverts to the inactive form. In contrast, in long-day conditions, hypocotyl elongation occurs during the daytime when phyB is in the photoactivated form. Warm temperatures can induce hypocotyl growth in both long-day and short-day conditions. However, the corresponding daytime and nighttime temperature responses reflect distinct underpinning mechanisms. Here, we describe assays for dissecting the mechanisms between daytime and nighttime thermoresponsive hypocotyl elongation.

Analysis of Phase Separation of EARLY FLOWERING 3.

Phase separation is an important mechanism for regulating various cellular functions. The EARLY FLOWERING 3 (ELF3) protein, an essential element of the EVENING COMPLEX (EC) involved in circadian clock regulation, has been shown to undergo phase separation. ELF3 is known to significantly influence elongation growth and flowering time regulation, and this is postulated to be due to whether the protein is in the dilute or phase-separated state. Here, we present a brief overview of methods for analyzing ELF3 phase separation in vitro, including the generation of phase diagrams as a function of pH and salt versus protein concentrations, optical microscopy, fluorescence recovery after photobleaching (FRAP), and turbidity assays.

Investigating Circadian Gating of Temperature Responsive Genes.

Understanding gene expression dynamics in the context of the time of day and temperature response is an important part of understanding plant thermotolerance in a changing climate. Performing "gating" experiments under constant conditions and light-dark cycles allows users to identify and dissect the contribution of the time of day and circadian clock to the dynamic nature of stress-responsive genes. Here, we describe the design of specific laboratory experiments in plants (Arabidopsis thaliana and bread wheat, Triticum aestivum) to investigate temporal responses to heat (1 h at 37 °C) or cold (3 h at 4 °C), and we include known marker genes that have circadian-gated responses to temperature changes.

Meal timing and its role in obesity and associated diseases.

Meal timing emerges as a crucial factor influencing metabolic health that can be explained by the tight interaction between the endogenous circadian clock and metabolic homeostasis. Mistimed food intake, such as delayed or nighttime consumption, leads to desynchronization of the internal circadian clock and is associated with an increased risk for obesity and associated metabolic disturbances such as type 2 diabetes and cardiovascular diseases. Conversely, meal timing aligned with cellular rhythms can optimize the performance of tissues and organs. In this review, we provide an overview of the metabolic effects of meal timing and discuss the underlying mechanisms. Additionally, we explore factors influencing meal timing, including internal determinants such as chronotype and genetics, as well as external influences like social factors, cultural aspects, and work schedules. This review could contribute to defining meal-timing-based recommendations for public health initiatives and developing guidelines for effective lifestyle modifications targeting the prevention and treatment of obesity and associated metabolic diseases. Furthermore, it sheds light on crucial factors that must be considered in the design of future food timing intervention trials.

Receptor-Mediated and Receptor-Independent Actions of Melatonin in Vertebrates.

Melatonin (N-acetyl-5-methoxytryptamine) is an indolamine that is synthesized from tryptophan in the pineal glands of vertebrates through four enzymatic reactions. Melatonin is a quite unique bioactive substance, characterized by a combination of both receptor-mediated and receptor-independent actions, which promote the diverse effects of melatonin. One of the main functions of melatonin, via its membrane receptors, is to regulate the circadian or seasonal rhythm. In mammals, light information, which controls melatonin synthesis, is received in the eye, and transmitted to the pineal gland, via the suprachiasmatic nucleus, where the central clock is located. Alternatively, in many vertebrates other than mammals, the pineal gland cells, which are involved in melatonin synthesis and secretion and in the circadian clock, directly receive light. Recently, it has been reported that melatonin possesses several metabolic functions, which involve bone and glucose, in addition to regulating the circadian rhythm. Melatonin improves bone strength by inhibiting osteoclast activity. It is also known to maintain brain activity during sleep by increasing glucose uptake at night, in an insulin-independent manner. Moreover, as a non-receptor-mediated action, melatonin has antioxidant properties. Melatonin has been proven to be a potent free radical scavenger and a broad-spectrum antioxidant, even protecting organisms against radiation from space. Melatonin is a ubiquitously distributed molecule and is found in bacteria, unicellular organisms, fungi, and plants. It is hypothesized that melatonin initially functioned as an antioxidant, then, in vertebrates, it combined this role with the ability to regulate rhythm and metabolism, via its receptors.

Circadian Regulation of Cardiac Arrhythmias and Electrophysiology.

Circadian rhythms in physiology and behavior are ≈24-hour biological cycles regulated by internal biological clocks (ie, circadian clocks) that optimize organismal homeostasis in response to predictable environmental changes. These clocks are present in virtually all cells in the body, including cardiomyocytes. Many decades ago, clinicians and researchers became interested in studying daily patterns of triggers for sudden cardiac death, the incidence of sudden cardiac death, and cardiac arrhythmias. This review highlights historical and contemporary studies examining the role of day/night rhythms in the timing of cardiovascular events, delves into changes in the timing of these events over the last few decades, and discusses cardiovascular disease-specific differences in the timing of cardiovascular events. The current understanding of the environmental, behavioral, and circadian mechanisms that regulate cardiac electrophysiology is examined with a focus on the circadian regulation of cardiac ion channels and ion channel regulatory genes. Understanding the contribution of environmental, behavioral, and circadian rhythms on arrhythmia susceptibility and the incidence of sudden cardiac death will be essential in developing future chronotherapies.

Circadian Influences on Myocardial Ischemia-Reperfusion Injury and Heart Failure.

The impact of circadian rhythms on cardiovascular function and disease development is well established, with numerous studies in genetically modified animals emphasizing the circadian molecular clock's significance in the pathogenesis and pathophysiology of myocardial ischemia and heart failure progression. However, translational preclinical studies targeting the heart's circadian biology are just now emerging and are leading to the development of a novel field of medicine termed circadian medicine. In this review, we explore circadian molecular mechanisms and novel therapies, including (1) intense light, (2) small molecules modulating the circadian mechanism, and (3) chronotherapies such as cardiovascular drugs and meal timings. These promise significant clinical translation in circadian medicine for cardiovascular disease. (4) Additionally, we address the differential functioning of the circadian mechanism in males versus females, emphasizing the consideration of biological sex, gender, and aging in circadian therapies for cardiovascular disease.

Circadian Rhythms in Cardiovascular Metabolism.

Energetic demand and nutrient supply fluctuate as a function of time-of-day, in alignment with sleep-wake and fasting-feeding cycles. These daily rhythms are mirrored by 24-hour oscillations in numerous cardiovascular functional parameters, including blood pressure, heart rate, and myocardial contractility. It is, therefore, not surprising that metabolic processes also fluctuate over the course of the day, to ensure temporal needs for ATP, building blocks, and metabolism-based signaling molecules are met. What has become increasingly clear is that in addition to classic signal-response coupling (termed reactionary mechanisms), cardiovascular-relevant cells use autonomous circadian clocks to temporally orchestrate metabolic pathways in preparation for predicted stimuli/stresses (termed anticipatory mechanisms). Here, we review current knowledge regarding circadian regulation of metabolism, how metabolic rhythms are synchronized with cardiovascular function, and whether circadian misalignment/disruption of metabolic processes contribute toward the pathogenesis of cardiovascular disease.

Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

Stroke in the Time of Circadian Medicine.

Time-of-day significantly influences the severity and incidence of stroke. Evidence has emerged not only for circadian governance over stroke risk factors, but also for important determinants of clinical outcome. In this review, we provide a comprehensive overview of the interplay between chronobiology and cerebrovascular disease. We discuss circadian regulation of pathophysiological mechanisms underlying stroke onset or tolerance as well as in vascular dementia. This includes cell death mechanisms, metabolism, mitochondrial function, and inflammation/immunity. Furthermore, we present clinical evidence supporting the link between disrupted circadian rhythms and increased susceptibility to stroke and dementia. We propose that circadian regulation of biochemical and physiological pathways in the brain increase susceptibility to damage after stroke in sleep and attenuate treatment effectiveness during the active phase. This review underscores the importance of considering circadian biology for understanding the pathology and treatment choice for stroke and vascular dementia and speculates that considering a patient's chronotype may be an important factor in developing precision treatment following stroke.

The role of temperature on the development of circadian rhythms in honey bee workers.

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

Hypoxic Conditions Modulate Chondrogenesis through the Circadian Clock: The Role of Hypoxia-Inducible Factor-1α.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor composed of an alpha and a beta subunit. HIF-1α is a master regulator of cellular response to hypoxia by activating the transcription of genes that facilitate metabolic adaptation to hypoxia. Since chondrocytes in mature articular cartilage reside in a hypoxic environment, HIF-1α plays an important role in chondrogenesis and in the physiological lifecycle of articular cartilage. Accumulating evidence suggests interactions between the HIF pathways and the circadian clock. The circadian clock is an emerging regulator in both developing and mature chondrocytes. However, how circadian rhythm is established during the early steps of cartilage formation and through what signaling pathways it promotes the healthy chondrocyte phenotype is still not entirely known. This narrative review aims to deliver a concise analysis of the existing understanding of the dynamic interplay between HIF-1α and the molecular clock in chondrocytes, in states of both health and disease, while also incorporating creative interpretations. We explore diverse hypotheses regarding the intricate interactions among these pathways and propose relevant therapeutic strategies for cartilage disorders such as osteoarthritis.

Autophagy as a new player in the regulation of clock neurons physiology of Drosophila melanogaster.

Axonal terminals of the small ventral lateral neurons (sLNvs), the circadian clock neurons of Drosophila, show daily changes in their arborization complexity, with many branches in the morning and their shrinkage during the night. This complex phenomenon is precisely regulated by several mechanisms. In the present study we describe that one of them is autophagy, a self-degradative process, also involved in changes of cell membrane size and shape. Our results showed that autophagosome formation and processing in PDF-expressing neurons (both sLNv and lLNv) are rhythmic and they have different patterns in the cell bodies and terminals. These rhythmic changes in the autophagy activity seem to be important for neuronal plasticity. We found that autophagosome cargos are different during the day and night, and more proteins involved in membrane remodeling are present in autophagosomes in the morning. In addition, we described for the first time that Atg8-positive vesicles are also present outside the sLNv terminals, which suggests that secretory autophagy might be involved in regulating the clock signaling network. Our data indicate that rhythmic autophagy in clock neurons affect the pacemaker function, through remodeling of terminal membrane and secretion of specific proteins from sLNvs.

YTHDF1-CLOCK axis contributes to pathogenesis of allergic airway inflammation through LLPS.

N6-methyladenosine (m6A) modification has been implicated in many cell processes and diseases. YTHDF1, a translation-facilitating m6A reader, has not been previously shown to be related to allergic airway inflammation. Here, we report that YTHDF1 is highly expressed in allergic airway epithelial cells and asthmatic patients and that it influences proinflammatory responses. CLOCK, a subunit of the circadian clock pathway, is the direct target of YTHDF1. YTHDF1 augments CLOCK translation in an m6A-dependent manner. Allergens enhance the liquid-liquid phase separation (LLPS) of YTHDF1 and drive the formation of a complex comprising dimeric YTHDF1 and CLOCK mRNA, which is distributed to stress granules. Moreover, YTHDF1 strongly activates NLRP3 inflammasome production and interleukin-1ß secretion leading to airway inflammatory responses, but these phenotypes are abolished by deleting CLOCK. These findings demonstrate that YTHDF1 is an important regulator of asthmatic airway inflammation, suggesting a potential therapeutic target for allergic airway inflammation.