Search Results (186)

Melatonin, a methoxyindole synthesized by the pineal gland, is secreted in response to photoperiodic cues relayed from the retina through an endogenous circadian oscillator within the suprachiasmatic nucleus. Consequently, melatonin secretion regulates the circadian rhythm. Melatonin has been reported to have antioxidant, photoprotective, anti-inflammatory, anticancer, and wound-healing properties. It has also been reported to promote hair growth, although the underlying mechanisms remain unclear. In this study, we explored the potential molecular mechanisms of melatonin-induced hair growth by using an in vivo C57BL/6 mouse model. We observed morphological changes in the dorsal area and changes in the hair cycle and anagen induction were observed through hematoxylin–eosin staining. The molecular mechanisms were explored using Western blotting and immunofluorescence assay. Our findings indicate that topical melatonin promotes anagen entry and hair regrowth in C57BL/6 mice, accompanied by the modulation of Wnt/β-catenin-related signaling proteins. The decrease in grayscale value, increase in hair length and skin thickness and histological change in hair follicles in the melatonin-treated group indicated hair regrowth in the dorsal skin of mice. Moreover, the expression of the Wnt/β-catenin pathway was remarkably regulated. These findings further our understanding of the molecular mechanisms underlying topical melatonin-induced hair growth. Full article

Intermittent fasting (IF) has gained increasing attention as a dietary strategy to improve metabolic health and prevent cardiometabolic disorders. Accumulating evidence suggests that modulation of the gut microbiota may represent one of the mechanisms underlying the physiological benefits of IF. This review summarizes the current knowledge on the mechanisms by which IF modulates gut microbial ecology and how these changes influence host metabolic and immune functions. We examine the effects of IF on gut microbiota diversity and composition, highlighting shifts in key microbial taxa associated with metabolic regulation. In addition, we discuss how fasting-induced microbial remodeling affects microbiota-derived metabolites, including short-chain fatty acids and bile acids, which play central roles in energy homeostasis, intestinal barrier integrity, and inflammatory signaling. Increasing evidence indicates that IF interacts with circadian rhythms, influencing both microbial oscillations and host metabolic pathways that coordinate nutrient sensing and energy metabolism. Furthermore, we explore the bidirectional crosstalk between the gut microbiota and the intestinal immune system, emphasizing that fasting-driven microbial changes may modulate inflammatory responses, epithelial barrier function, and immune cell activity. Finally, we discuss nutritional strategies that may enhance the beneficial effects of IF, including the incorporation of prebiotics, dietary fiber, and probiotic supplementation, to promote microbial diversity and functional resilience. Collectively, these findings support a model in which IF acts as a key modulator of the gut microbiota–immune–metabolic axis. Future integrative studies combining gut microbiome, metabolomic, and immunological approaches are needed to better understand these interactions and optimize microbiota-targeted dietary interventions. Full article

Parkinson’s disease (PD) is a progressive neurodegenerative disorder in which circadian rhythm disruption (CRD) emerges as both a prodromal feature and a potential pathogenic driver. Elucidating the bidirectional interplay between PD and CRD is essential for identifying early biomarkers and developing chronotherapeutic strategies. We narratively synthesized literature published over the past two decades in PubMed, Web of Science, and CNKI, focusing on molecular mechanisms, clinical manifestations, biomarker development, and interventional studies addressing the PD–CRD interface. In the CRD-PD direction, circadian disruption accelerates dopaminergic neurodegeneration through four convergent mechanisms: (i) REV-ERBα–mediated dysregulation of dopamine biosynthesis and NF-κB/NLRP3-driven neuroinflammation; (ii) impaired sleep-dependent glymphatic clearance of α-synuclein (α-syn); (iii) NAD⁺–SIRT1–BMAL1–PGC-1α axis dysfunction leading to mitochondrial bioenergetic failure; and (iv) C/EBPβ-dependent autophagic rhythm disruption coupled with pro-inflammatory microglial activation, collectively establishing a dual pro-inflammatory–autophagy-suppressive milieu permissive for α-syn aggregation. In the reverse PD-CRD direction, PD pathology destabilizes the circadian system via Braak-stage degeneration of rhythm-regulatory nuclei, retinal dopaminergic denervation attenuating SCN photic entrainment, pineal–melatonin axis suppression, iatrogenic effects of dopaminergic pharmacotherapy, and gut microbiota dysbiosis propagated through the microbiota–gut–brain axis. Emerging multi-modal chronobiomarkers—including peripheral clock gene expression profiles, melatonin secretion patterns, tryptophan–kynurenine metabolites, and gut microbial oscillation signatures—show promise for prodromal diagnosis and disease subtyping. Circadian-targeted precision interventions—encompassing timed bright light therapy, exogenous melatonin, and chronopharmacological interventions—represent a promising translational paradigm for the early identification and management of PD. Full article

Earth’s rotation produces day and night cycles that are a primary selective pressure driving the evolution of endogenous circadian clocks. Cyanobacteria are the most studied prokaryotic model, and their timekeeping core is a protein oscillator composed of KaiA, KaiB, and KaiC. This oscillator sustains a near-24 h rhythm independently of transcription–translation feedback, challenging the long-standing assumption that prokaryotes merely respond passively to environmental cues. Moreover, it offers unique insights into the evolution and operational logic of circadian clocks. This review summarizes advances in cyanobacterial circadian research. We first analyze the KaiABC oscillator’s molecular basis, including synergistic conformational changes, phosphorylation and dephosphorylation cascades, and temperature compensation, which confer robustness and tunability. We compare oscillator compositions across cyanobacterial lineages, showing evolutionary plasticity. We then outline input and output networks, clarifying how environmental signals reset the oscillator phase and how temporal information is relayed to downstream processes. We further explain how the clock coordinates photosynthesis, nitrogen fixation, respiration, and cell division through predictive regulation, temporal decoupling, and resource prioritization, thereby resolving metabolic conflicts and enhancing fitness under light and dark cycles. This framework provides a theoretical basis for microbial survival strategies in fluctuating environments and offers insights for synthetic biology circuit design. Finally, we discuss open questions, including coupling between the oscillator and the cell cycle, functional divergence among ecotypes, and roles at the community level. Further research on the cyanobacterial clock will help clarify general principles of biological timing and its evolutionary origins. Full article

Circadian rhythm disruptions are increasingly recognized in disorders such as obstructive sleep apnea (OSA), yet analysis of 24 h gene expression patterns remains challenging due to the lack of reliable reference genes for normalization. Even commonly used housekeeping genes may exhibit circadian oscillations, which can confound rhythmic gene expression analyses and hinder biomarker identification. To address this limitation, we evaluated the gene expression stability of 11 commonly used housekeeping genes in blood collected every 6 h over 24 h period from 40 adults with varying OSA severity and controls. Stability ranking by analytical tools RefFinder and EndoGeneAnalyzer identified ACTB (β-actin) and RPL13A (ribosomal protein L13a) as the most consistent reference genes, with minimal intra- and inter-individual variability across sampling times and disease groups. Their suitability was assessed by personalized cosinor analysis of core clock genes (BMAL1, PER2, CRY1), demonstrating that appropriate normalization enables detection of circadian oscillations in clinical samples. Using the optimal normalization, CosinorPy analysis of the core clock genes revealed significant circadian oscillations of at least one clock gene in the studied participants. These findings establish ACTB and RPL13A as robust reference genes for blood-based circadian studies of OSA and provide an important methodological framework for future circadian biomarker research. Full article

The circadian rhythm is an internal timing system formed by the body’s adaptation to the Earth’s rotation, which helps maintain homeostasis by regulating physiological, metabolic, and behavioral activities. The gut microbiota (GM), the largest microbial ecosystem in the human body, exhibits a bidirectional regulatory relationship with the host circadian clock. Emerging evidence indicates that circadian rhythm disruption (CRD) is linked to disturbances in the diurnal oscillations and compositional balance of the GM, accompanied by reduced short-chain fatty acid levels, increased lipopolysaccharide leakage, and altered tryptophan metabolism. These microbial abnormalities may be involved in anxiety-like behaviors through three major pathways: neuroendocrine (hyperactivation of the HPA axis), immune (microglia-mediated neuroinflammation), and neurotransmitter (imbalance of the serotonergic and dopaminergic systems). Conversely, microbial metabolites such as butyrate and secondary bile acids may reciprocally regulate peripheral clock gene expression, forming a complex “circadian rhythm–GM–anxiety” interaction network. This review summarizes the molecular basis of circadian–GM interactions, potential GM-mediated mechanisms linking CRD with anxiety, and emerging intervention strategies including chrononutrition (time-restricted feeding, sequential nutrient intake), microbiota-targeted therapies (probiotics/prebiotics, fecal microbiota transplantation), and light therapy and melatonin supplementation. Future directions should focus on cell-specific mechanisms using single-cell and spatial transcriptomics, developing personalized interventions that integrate chronotype and microbiome profiling, and conducting large-scale randomized controlled trials to facilitate clinical translation. This review provides a framework for understanding the integrative role of circadian biology and gut microbiota in anxiety and may help develop precision intervention paradigms. Full article

Photobiomodulation (PBM) is a non-invasive therapeutic strategy that uses red and near-infrared (NIR) light in the 590–950 nm range to modulate the cellular and molecular pathways involved in retinal homeostasis. At the molecular level, PBM acts primarily through photon absorption by cytochrome c oxidase (CcO, complex IV of the mitochondrial electron transport chain), whose four metal centres—two copper (CuA and CuB) and two heme groups (heme a and heme a₃)—absorb light across approximately 600–1000 nm. Photon capture promotes photodissociation of inhibitory nitric oxide (NO) from the binuclear CuB–heme a₃ centre, accelerates electron transfer, restores the proton-motive force and increases ATP synthesis. These primary events trigger a coordinated molecular programme that includes (i) transient mitochondrial reactive oxygen species (ROS) bursts that activate the Nrf2/Keap1/ARE axis and upregulate phase II antioxidant enzymes (HO-1, NQO1, GCLC, SOD2, catalase, GPx); (ii) calcium- and cAMP-dependent secondary signalling that converges on PI3K/Akt, MAPK/ERK, AMPK and mTOR pathways; (iii) suppression of NF-κB-driven cytokine production (TNF-α, IL-1β, IL-6) and of NLRP3 inflammasome activation; (iv) downregulation of the HIF-1α/VEGF axis, particularly at 590 nm; (v) anti-apoptotic remodelling of the Bcl-2/Bax ratio with reduced cytochrome c release and caspase-3/9 activation; and (vi) PGC-1α/TFAM/NRF1-driven mitochondrial biogenesis, alongside restoration of fission/fusion homeostasis (Drp1, Mfn1/2, Opa1) and PINK1/Parkin-mediated mitophagy. Wavelength specificity has a defined molecular basis: 590 nm modulates VEGF signalling and RPE pump activity, 660 nm interacts with the CuB centre and enhances O₂ binding at CcO, and 850 nm is absorbed by CuA and supports electron entry into complex IV. A second molecular axis is the bidirectional crosstalk between PBM and the circadian system: mitochondrial respiration, ATP turnover and CcO activity oscillate over the 24 h cycle under the control of the BMAL1/CLOCK and PER/CRY core machinery, the NAD⁺/SIRT1–SIRT3 axis and REV-ERBα. Preliminary preclinical and human observations suggest that NIR-induced bioenergetic and functional gains may be coupled to this rhythm, with greater benefit reported when light is delivered in the morning window (≈08:00–11:00); this time dependence should be regarded as an emerging hypothesis rather than an established clinical principle. The clinical evidence is unevenly developed across indications. It is most robust for non-exudative age-related macular degeneration, where multiwavelength PBM (590/660/850 nm; Valeda Light Delivery System) has shown disease-modifying potential in randomized controlled trials (LIGHTSITE I–III and the LIGHTSITE IIIB extension), with sustained BCVA gains and reduced incidence of geographic atrophy over 24 months and beyond. Evidence for retinitis pigmentosa, central serous chorioretinopathy and, with red-light monotherapy, childhood myopia is at present limited to small or short-term studies and remains preliminary. This narrative review synthesizes the molecular machinery engaged by PBM, integrates clinical findings across retinal diseases and discusses how chronotherapeutic delivery of light, aligned with the molecular clock, may further optimize therapeutic efficacy. Full article

Circadian rhythms impose temporal organization on immune function, shaping host responses to infection, injury, and chronic disease. While transcriptional control by core clock components such as CLOCK and BMAL1 has been extensively characterized, this paradigm alone cannot explain the rapid and dynamic nature of immune signaling. Emerging evidence identifies post-translational modifications (PTMs)—including phosphorylation, ubiquitination, and acetylation—as critical regulators that confer speed, reversibility, and specificity to inflammatory pathways. Here, we propose the concept of a “Chrono-PTM axis,” in which circadian timing and PTM-dependent signaling are functionally integrated to govern immune activation thresholds. We discuss how PTMs not only regulate core clock machinery but also temporally gate key innate immune pathways, including NF-κB signaling and inflammasome activation, thereby controlling cytokine production at multiple levels. Furthermore, we highlight the role of immunometabolism in supplying essential cofactors that couple cellular energetic states to PTM dynamics, linking metabolic oscillations to inflammatory outputs. Disruption of this axis contributes to the pathogenesis of autoimmune diseases, cancer, and tissue-specific inflammatory disorders. Finally, we outline emerging therapeutic opportunities targeting the Chrono-PTM axis, including chronotherapy and PTM-directed interventions, and identify critical gaps in temporal proteomics and translational studies. Elucidating the integration of circadian and post-translational regulation will provide a unifying framework for understanding immune homeostasis and may enable time-informed precision immunotherapy. Full article

The circadian clock is closely linked to glucose regulation, but the dynamical consequences of specific metabolic feedback pathways on core clock regulation remain incompletely understood. In this study, we developed a theoretical metabolic-circadian model incorporating a REV-ERB$\mathrm{\alpha }$-glucagon-glucose feedback pathway. The model extends a previously established mammalian circadian clock framework by introducing glucagon-mediated regulation of blood glucose and glucose-dependent modulation of Rev-erbα transcription. Using this model, we examined how the feedback pathway affects circadian oscillations, the sensitivity of period and amplitude to parameter perturbations, and phase-related responses under light stimulation and light–dark cycles. Simulations of the feedback-related parameters showed that the glucose-to-clock feedback strength had a marked effect on oscillation period and amplitude, motivating a further assessment of whether regular circadian dynamics were preserved under parameter perturbations. We therefore analyzed both one-parameter perturbations and simultaneous perturbations of all model parameters. For one-parameter scans, we quantified not only the oscillatory boundaries but also the period variation and the amplitude variation of Per and Rev-erbα within the oscillatory ranges. For simultaneous all-parameter perturbations, Latin hypercube sampling was used to compare coupled and uncoupled models under bounded perturbation ranges. The coupled model showed a higher fraction of regular circadian oscillations under local perturbations, mainly by reducing the probability of rhythm loss. We further examined phase responses and light–dark entrainment to assess how the feedback affects dynamical properties beyond period and amplitude. In the phase-response analysis, the feedback reduced excessive phase shifts in the model, suggesting a possible phase-response robustness effect in this theoretical framework. These theoretical results suggest that the REV-ERB$\mathrm{\alpha }$-glucagon-glucose feedback pathway may be relevant to circadian regulation under fasting-associated metabolic conditions. Full article

Growing evidence indicates that the gut microbiota is not a static ecosystem but a rhythmic metabolic organ whose oscillatory activity is tightly coordinated with host circadian biology. Disruption of this temporal alignment, through irregular diet, sleep disturbance, shift work, or social jet lag, may profoundly alter microbial composition and the production of neuroactive metabolites. These alterations have emerged as potential contributors to the pathophysiology of mood disorders. This review introduces the concept of chrono-metabolic psychiatry, a framework integrating circadian rhythms, gut microbiota dynamics, and host metabolic signaling in the development and course of depressive and bipolar disorders. In this framework, the term “chrono-metabolic” refers to the integration of biological timing, host metabolic regulation, and microbiota-derived metabolic signaling. Chrono-metabolic psychiatry therefore shifts the focus from static dysbiosis or neurotransmitter imbalance alone to the time-dependent interactions among circadian misalignment, microbial rhythmicity, immune regulation, metabolite production, and affective instability. Diurnal fluctuations in short-chain fatty acids, tryptophan–kynurenine metabolites, bile acids, and microbial-derived neurotransmitters interact with clock gene regulation, hypothalamic–pituitary–adrenal axis activity, neuroinflammation, and synaptic plasticity. Chrono-disruption may represent a transdiagnostic vulnerability factor and may confirm the bidirectional relationship between mood instability and microbiota rhythmicity. Emerging therapeutic implications, including chrono-nutrition, time-restricted feeding, targeted probiotic administration (“chronobiotics”), and the microbiota-modulating effects of psychotropic medications are discussed. By shifting from a compositional to a temporal–metabolic perspective, this model highlights the importance of microbial oscillations rather than static dysbiosis alone. Integrating circadian biology into microbiota research may enable metabolomic stratification and pave the way for precision psychiatry approaches grounded in host–microbe metabolic crosstalk. Future longitudinal and time-resolved multi-omics studies are needed to validate this framework and to translate it into clinically actionable interventions. Full article

The circadian clock is a widespread rhythmic phenomenon across organisms, characterized by distinct gene expression patterns and behaviors at specific times of the day. Extensive genetic studies in the model fungus Neurospora crassa have yielded critical insights into the components and molecular mechanisms of circadian oscillators. However, these understandings remain absent across fungal lineages, especially from edible mushrooms. Morels (Morchella spp.) are well-recognized edible ascomycetes of considerable economic value and are partially artificially cultivated, but their biological characteristics are poorly understood. Investigating the presence of their circadian clock components, as well as the molecular underpinnings of circadian rhythms, holds important biological implications. In this study, we firstly performed a genomic search for homologs of known circadian clock genes in Morchella sextelata. Homologs of seven circadian clock genes, including wc-1, wc-2, fwd-1, frh, frq, and two additional clock-controlled genes, were identified, indicating the components necessary for the operation of a FWC oscillator contained in M. sextelata. Then, using reverse transcription quantitative PCR (RT-qPCR), the expression profiles of these seven circadian clock-related genes and four mating-type genes were examined in RNA samples which were extracted from mycelia of MAT1-1, MAT1-2 and MAT1-1 × MAT1-2 co-culture/crossed condition during conidiation under in vitro cultivation across one day. The expression levels of seven circadian clock genes and four mating-type genes displayed similar time-of-day-specific rhythmic patterns, yet remained consistently distinct across the mating-type strains and their co-culture/crossed condition, indicating a potential correlation between circadian clock and mating-type loci. Collectively, these results suggest that M. sextelata harbors conserved circadian clock-related homologs and displays mating-type-associated temporal expression differences under the tested conidiation conditions, offering a novel perspective for exploring the potential link between clock-related regulation and mating-type background in the future. Full article

A high-fat (HF) diet disrupts metabolic homeostasis and impairs peripheral circadian rhythms in key metabolic tissues. β-Hydroxybutyrate (BHB), a major circulating ketone body, functions not only as an energy substrate but also as a signaling metabolite regulating nutrient-sensing and inflammatory pathways. However, its role in modulating metabolic–circadian interactions under conditions of nutrient excess remains unclear. In this study, we investigated whether BHB supplementation influences metabolic signaling and circadian clock oscillations in liver, skeletal muscle and adipose tissue under chow and HF conditions. Male C57BL/6 mice were fed chow or HF with or without BHB supplementation (500 mg/kg body weight in the diet) for 7 weeks. Metabolic parameters were assessed by indirect calorimetry, and tissues were collected every 4 h across the circadian cycle. HF feeding increased body weight and adiposity (p < 0.01), reduced AMPK activation, enhanced AKT/mTOR signaling, elevated NF-κB levels and dampened clock gene rhythmicity. BHB supplementation significantly decreased food intake in HF-fed mice (p < 0.01) and partially reversed several molecular alterations in a tissue-specific manner. In skeletal muscle and adipose tissue, BHB increased AMPK activation and reduced mTOR and NF-κB signaling (p < 0.05), whereas hepatic effects were more modest. Notably, BHB modulated circadian gene expression, restoring aspects of rhythmic amplitude and/or phase, particularly in adipose tissue. These findings may indicate that BHB supplementation modulates nutrient-sensing pathways and partially restores peripheral circadian rhythms under HF conditions. While some effects may be influenced by reduced energy intake, BHB may serve as a metabolic signal linking nutrient status to circadian regulation. Full article

Oxidative alcohol metabolism in the liver relies on sequential enzymatic reactions involving alcohol dehydrogenase (ADH), cytochrome P450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) isozymes. However, the circadian regulation of these enzymes, their susceptibility to genetic, environmental, and metabolic disruption, and their functional implications toward alcohol-mediated tissue injury remain incompletely defined. To address this gap, we performed a comprehensive integrative analysis of the publicly available circadian transcriptome datasets spanning genetic clock disruption, acute sleep deprivation, chronic high-fat diet feeding, and occupational shift work to systematically characterize the temporal regulation and disruption vulnerability of the major alcohol-metabolizing enzymes. Mouse tissue-cycling analyses revealed pronounced gene- and tissue-specific diurnal regulation, with Adh1 oscillating primarily in adipose tissues; Cyp2e1 and mitochondrial Aldh2 cycling broadly across kidney, aorta, lung, adrenal gland, and liver; and cytosolic Aldh1b1 being uniformly arrhythmic. In the liver, Cyp2e1 and Aldh2 exhibited robust ~24 h oscillations that peaked during the light/resting phase, while Adh1 showed inconsistent rhythmicity and Aldh1b1 remained arrhythmic. Notably, Cyp2e1 and Aldh2 rhythms persisted in Bmal1 knockout and Clock mutant livers under light–dark conditions, despite complete loss of core clock gene oscillations, yet were abolished in constant darkness, revealing that systemic zeitgeber cues can mask the loss of intrinsic clock function to maintain apparent rhythmicity in these metabolic genes. Systematic cross-paradigm comparison established a novel gene-specific vulnerability hierarchy. Aldh2 was found to be most disrupted by environmental and metabolic perturbations, with acute sleep deprivation eliminating its rhythmicity and temporal expression pattern and a Western-style high-fat diet inducing pronounced phase delays and rhythm loss relative to low-fat diet controls. Both disruptions paralleled alterations in hepatocyte nuclear factor 4α (Hnf4a), newly implicating HNF4α as a potential mediator of ALDH2 circadian instability. In humans, ALDH2 and CYP2E1 exhibited conserved but phase-inverted circadian rhythms across multiple tissues relative to mice, and, importantly, night-shift workers showed markedly dampened and phase-shifted ALDH2 rhythms in peripheral blood mononuclear cells, providing the molecular link between occupational circadian misalignment and impaired acetaldehyde detoxification. Collectively, our detailed and innovative analytical approach reveals gene- and tissue-specific circadian regulation of alcohol-metabolizing enzymes, identifies ALDH2 as uniquely vulnerable to circadian misalignment, underscores the importance of circadian timing for optimal hepatic detoxification and resistance to tissue injury, and suggests that monitoring circadian rhythms could help tailor individualized advice on alcohol consumption for shift workers and populations with irregular sleep schedules, informing precision medicine approaches for alcohol-related disorders. Full article

Turbot (Scophthalmus maximus) is an economically vital cold-water fish frequently challenged by summer heat and low salinity. However, the temporal response of physiological circadian rhythms to such long-term stress remains underexplored. This study investigated antioxidant, immune, and endocrine rhythms in turbot acclimated to control (16 °C, 30 ppt), high-temperature (23 °C), and low-salinity (10 ppt) conditions for 30 days. Subsequently, time-series sampling was performed every 4 h for 72 consecutive hours. Under optimal conditions, hepatic superoxide dismutase (SOD), serum alanine aminotransferase (ALT), and melatonin exhibited robust 24 h rhythms. Long-term stress disrupted this homeostasis through divergent mechanisms. Low-salinity stress induced “rhythmic remodeling,” maintaining balance via phase shifts or novel infradian (48–72 h) oscillations in thyroid hormones (T3, T4) and ALT, without oxidative damage. Conversely, high-temperature stress triggered “rhythmic collapse,” characterized by a loss of daily rhythms in SOD and ALT, sustained inflammation indicated by elevated acid phosphatase (ACP), metabolic depression (suppressed T3), and malondialdehyde accumulation. These findings demonstrate that heat stress poses a more destructive threat to circadian integrity than hyposmotic stress. Consequently, the rhythmic dynamics of ACP, ALT, T3, and T4 are identified as critical indicators of stress status, serving as potential biomarkers for screening stress-tolerant strains for selective breeding. Full article

Plants defend against pathogens such as fungi by initiating coordinated structural and chemical responses. Pathogen perception triggers rapid cytosolic calcium influx and calcium oscillations that drive defense gene expression, yet the mechanisms by which these signals encode stressor intensity and propagate systematically remain unclear. Here, we present a microfluidic system to characterize intracellular calcium dynamics in protonemal colonies of the moss Physcomitrium patens (Hedw.) upon precise and reversible exposure to fungal chitin oligosaccharides. Epifluorescent imaging of cells expressing the calcium indicator GCaMP6f revealed a rapid, coordinated calcium response to chitin addition, followed by stereotyped oscillations that subsided quickly upon stimulus removal. We implemented an unbiased image segmentation algorithm using pixel-based k-means clustering to automatically locate regions with specific oscillatory signatures. Calcium dynamics were distinct across adjacent cells, distinguishable by cell type, and significantly modulated by circadian rhythm, adaptation time within the device, and stimulus timing. Cytosolic calcium oscillations, which rose and fell symmetrically within about 60 s, occurred spontaneously during the subjective night and following short adaptation periods. Chitin elicited strong oscillations with increased frequency, amplitude, and duration, and repeated pulses entrained regular, colony-wide oscillations at the stimulation interval. This study complements prior investigations of whole plant and growth tip dynamics and provides a quantitative framework to study calcium signaling in plants, including mechanisms of signal propagation and the role of oscillation frequency on gene expression. Full article