Modulation Of Circadian Rhythms Research Articles

Modulation of Cellular Circadian Rhythms by Secondary Metabolites of Lichens.

BackgroundMost mammalian cells harbor molecular circadian clocks that synchronize physiological functions with the 24-h day-night cycle. Disruption of circadian rhythms, through genetic or environmental changes, promotes the development of disorders like obesity, cardiovascular diseases, and cancer. At the cellular level, circadian, mitotic, and redox cycles are functionally coupled. Evernic (EA) and usnic acid (UA), two lichen secondary metabolites, show various pharmacological activities including anti-oxidative, anti-inflammatory, and neuroprotective action. All these effects have likewise been associated with a functional circadian clock.Hypothesis/PurposeTo test, if the lichen compounds EA and UA modulate circadian clock function at the cellular level.MethodsWe used three different cell lines and two circadian luminescence reporter systems for evaluating dose- and time-dependent effects of EA/UA treatment on cellular clock regulation at high temporal resolution. Output parameters studied were circadian luminescence rhythm period, amplitude, phase, and dampening rate.ResultsBoth compounds had marked effects on clock rhythm amplitudes and dampening independent of cell type, with UA generally showing a higher efficiency than EA. Only in fibroblast cells, significant effects on clock period were observed for UA treated cells showing shorter and EA treated cells showing longer period lengths. Transient treatment of mouse embryonic fibroblasts at different phases had only minor clock resetting effects for both compounds.ConclusionSecondary metabolites of lichen alter cellular circadian clocks through amplitude reduction and increased rhythm dampening.

Effect of a hyaluronic acid-based mesotherapeutic injectable on the gene expression of CLOCK and Klotho proteins, and environmentally induced oxidative stress in human skin cells.

Normal circadian rhythms are essential to the repair mechanisms of oxidative stress implicated in skin aging. Given reports that hyaluronic acid (HA) homeostasis exhibits a different profile in chronological skin aging, as compared to environmental or extrinsic aging, an improved understanding of the way HA interacts with its surroundings, and the impact of HA injectables in replacing lost HA and encouraging rejuvenation, is of key benefit to skin aging treatments. The objectives of these current studies were twofold. Firstly, to demonstrate the in vitro effects of two lightweight hyaluronic-based injectables on the expression of CLOCK protein in human skin fibroblasts, and their effects on Klotho protein expression as a marker for circadian rhythms in a combined human keratinocyte and Merkel cell model. Secondly, to ascertain whether these findings could be correlated with in vitro effects on various environmental oxidative stress aging markers (blue light, UVA/UVB, Urban Dust, and IR exposures). Oxidative stress studies were aimed to highlight possible protective effects through different challenge conditions in two models, ex vivo human skin explants and in vitro monolayer cultures of normal human dermal fibroblasts (NHDF). The protective effects of the test products were evaluated against an increase of cyclobutene pyrimidine dimers (CPDs) abundance within epidermal section of ex vivo skin explants after UVA/UVB radiation; effects of blue light on gene expression from NHDFs fibroblasts; effects of pollutants (Urban dust, UbD) on gene expression in NHDFs fibroblasts; and an increase of reactive oxygen species (ROS) production by NHDFs fibroblasts after infrared-A radiation. Gene expression was assayed and analyzed utilizing microfluidic TaqMan qPCR arrays. CLOCK expression was measured in young and senescing NHDFs by immunostaining, and Klotho and melatonin expression by immunostaining in Merkel cell-enriched normal adult human epidermal cell cultures. In an aging culture of mixed keratinocyte and Merkel skin cells, activation of Klotho expression was induced by the application of both HA test products. Moreover, the HA products increase Klotho protein expression in both Merkel cells and keratinocytes. The observed positive effect of the tested products on melatonin receptors 1A and 1B expression in aging Merkel cell culture and keratinocytes is also interesting. HA-Y (developed for patients 25+ years old) stimulated melatonin receptors type 1B expression in aging cell cultures more strongly than HA-S (developed for patients 35-65 years old). In age (stressed) cells, a lower expression of Klotho protein and melatonin receptors 1A and 1B is apparent. The addition of HA-Y and HA-S stimulates their expression thus providing a "protective" effect. The blue light irradiation at 40 J/cm2 performed in NHDF fibroblast cultures led to a modification of the expression of several genes, all involved in mechanisms known to be modulated in case of solar radiation stress. Although these are preliminary findings, they are the first we know of that demonstrate HA facial injectables having a benefit and possibilities beyond the "physical filling" of the skin. As regards the beneficial effects against blue light-induced oxidative stress, and a return to cellular homeostasis, there is a need to conduct further and more precise investigations into HA-S. Furthermore, the benefit of these HA injectables (Novacutan®) in the modulation of oxidative stressed circadian rhythms widens their potential benefit.

Circadian molecular clock disruption in chronic pulmonary diseases.

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Light, sleep-wake rhythm, and behavioural and psychological symptoms of dementia in care home patients: Revisiting the sundowning syndrome.

ObjectivesIt is believed that inadequate environmental light, especially in facilities such as care homes, contribute to the diurnal changes of behavioural and psychological symptoms of dementia (BPSD) historically referred to as “sundowning syndrome”. Conceptual models of sundowning phenomena have shifted emphasis from the role of light in vision (image forming) to its role in circadian rhythm modulation. However, the grounds for this change are unclear and the evidence on which it is based has not been examined comprehensively.MethodsWe have searched literature on sundowning syndrome and its association with light and studies evaluating BPSD, behavioural rhythm and environmental light in care homes in four databases (PubMed, Web of Science, Embase and Cochrane Library) from inception to 31 January 2021.ResultsOf the nine studies investigating light, behavioural rhythm and BPSD in care homes identified, we found evidence that insufficient natural light exposure was associated with worsening of BPSD and disrupted activity rhythm but it was not clear whether this related to image forming or disrupted circadian rhythm. There was a paucity of evidence in relation to the role of low levels of light for image forming in the context of a specific BPSD symptom: visual hallucinations. We also found literature on the possible role of light outside the visible spectrum influencing cognition. Based on the evidence, we proposed a new model integrating different components of light in BPSD and sundowning syndrome that combines its image forming and circadian roles.ConclusionsInadequate light may be a risk factor for BPSD and sundowning syndrome for dementia patients through a range of different mechanisms. It is recommended that multiple neuro‐endocrinological and socio‐environmental factors relevant to light such as adjusting the environmental setting, increasing light exposure, and scheduling activities should be considered when treating dementia patients with BPSD.

Review study on epileptiform discharge during sleep and circadian rhythm modulation during seizures

Epilepsy is a neurological condition that affects approximately more than 50 million people around the globe, out of which the prevalence of this disease is also seen in developed countries. Epilepsy can be clinical defined by two or more non triggered seizures. A seizure can be characterized as a paroxysmal event which is caused due to hyper–synchronization in the neurons that many have various reasons of manifestation. The neuronal discharge maybe focal spreading in specific brain cortex or widespread throughout the cortex. An Electroencephalogram (EEG) is used to in diagnosis and management of epilepsy and can be used for routine examinations, it also considered to be the golden standard investigation of epileptic syndrome. While using EEG the distribution, presence and frequency of interictal epileptiform discharges IEDs are suggestive diagnosis of epilepsy. In other words the presence of IEDs in an individual’s EEG is a diagnosis of epilepsy. IEDs are predominantly found in EEG that is performed during sleep which can give basic diagnosis and prognosis of sleep related epilepsies in patients. During sleep latent interictal discharge are activated. Sleep IEDs are said to greatly affect the epilepsy management. These interaction mechanisms of sleep and epilepsies tends to bring about changes in behaviour of the seizure.

Beneficial effects of melatonin on liver fibrosis: A systematic review of current biological evidence.

Hepatic fibrosis is a reversible response to either acute or chronic cellular injury from a wide variety of etiologies, characterized by excessive deposition of extracellular matrix resulting in liver dysfunction and cirrhosis. Melatonin (N‐acetyl‐5‐methoxytryptamine), the main product secreted by the pineal gland, is a multitasking indolamine with important physiological functions such as anti‐inflammatory and antioxidant actions, modulation of circadian rhythms, and immune system enhancement. Among the numerous biological activities of melatonin, its antifibrotic effects have received increasingly more attention. In this study, we performed a systematic review of publications of the last 10 years evaluating the mechanisms of action of melatonin against liver fibrosis. The study protocol was registered at PROSPERO (CRD42022304744). Literature research was performed employing PubMed, Scopus, and Web of Science (WOS) databases, and after screening, 29 articles were included. Results from the selected studies provided denoted the useful actions of melatonin on the development, progression, and evolution of liver fibrosis. Melatonin antifibrotic effects in the liver involved the reduction of profibrogenic markers and modulation of several cellular processes and molecular pathways, mainly acting as an antioxidant and anti‐inflammatory agent. In addition, the indolamine influenced different molecular processes, such as hepatocyte apoptosis, modulation of autophagy and mitophagy, restoration of circadian rhythms, and modulation of microRNAs, among others. Although some limitations have been found regarding variability in the study design, the findings here summarized display the potential role of melatonin in ameliorating the development of liver fibrosis and its possible progression to liver cirrhosis and hepatocarcinoma.

Wavelengths and irradiances modulate the circadian rhythm of Neurospora crassa.

The circadian rhythm affects the biological evolution and operating mechanisms of organisms. The impact of light on the circadian rhythm is a significant concern for both biology and human well-being. However, the relation between different wavelengths, irradiances, and circadian rhythm is unknown. In this study, we compared the effects of four different monochromatic light-emitting diode (LED) light and two different irradiances on the circadian rhythm of a wild-type Neurospora crassa. The results demonstrated that the circadian rhythm of Neurospora crassa can be modulated by violet (λp = 393 nm), blue (λp = 462 nm), and green (λp = 521 nm) light, regardless of the irradiances, in the visible region. Unexpectedly, for the yellow light (λp = 591 nm), the 2 W/m2 light had a more significant impact on circadian rhythm modulation than the 0.04 W/m2 light had. Considering the highest energy of yellow light (2.25 eV) is lower than the High Occupied Molecular Orbital (HOMO)-Lowest Unoccupied Molecular Orbital (LUMO) gap of WC-1 (2.43 eV). We speculate that there may be other potential photoreceptors that are involved in circadian rhythm modulation. The HOMO-LOMO gaps of these proteins are greater than 1.98 eV and less than 2.25 eV. These results provide a strong foundation for a deeper understanding of the impact of different light on the circadian rhythm and also shed light on the identification of new circadian rhythm modulation photoreceptors.

Recommendations for daytime, evening, and nighttime indoor light exposure to best support physiology, sleep, and wakefulness in healthy adults.

Ocular light exposure has important influences on human health and well-being through modulation of circadian rhythms and sleep, as well as neuroendocrine and cognitive functions. Prevailing patterns of light exposure do not optimally engage these actions for many individuals, but advances in our understanding of the underpinning mechanisms and emerging lighting technologies now present opportunities to adjust lighting to promote optimal physical and mental health and performance. A newly developed, international standard provides a SI-compliant way of quantifying the influence of light on the intrinsically photosensitive, melanopsin-expressing, retinal neurons that mediate these effects. The present report provides recommendations for lighting, based on an expert scientific consensus and expressed in an easily measured quantity (melanopic equivalent daylight illuminance (melaponic EDI)) defined within this standard. The recommendations are supported by detailed analysis of the sensitivity of human circadian, neuroendocrine, and alerting responses to ocular light and provide a straightforward framework to inform lighting design and practice.

Melatonin as an Antioxidant Agent in Stroke: An Updated Review.

Stroke is a devastating disease associated with high mortality and disability worldwide, and is generally classified as ischemic or hemorrhagic, which share certain similar pathophysiological processes. Oxidative stress is a critical factor involved in stroke-induced injury, which not only directly damages brain tissue, but also enhances a series of pathological signaling cascades, contributing to inflammation, brain edema, and neuronal death. To alleviate these serious secondary brain injuries, neuroprotective agents targeting oxidative stress inhibition may serve as a promising treatment strategy. Melatonin is a hormone secreted by the pineal gland, and has various properties, such as antioxidation, anti-inflammation, circadian rhythm modulation, and promotion of tissue regeneration. Numerous animal experiments studying stroke have confirmed that melatonin exerts considerable neuroprotective effects, partially via anti-oxidative stress. In this review, we introduce the possible role of melatonin as an antioxidant in the treatment of stroke based on the latest published studies of animal experiments and clinical research.

Expression of CRY2 Gene in the Brain Is Related to Human Navigation.

Navigation is a complex cognitive process. CRY2 gene has been proposed to play an important role in navigation behaviors in various non-human animal species. Utilizing a recently developed neuroimaging-transcriptomics approach, the present study reported a tentative link between the CRY2 gene and human navigation. Specifically, we showed a significant pattern similarity between CRY2 gene expression in the human brain and navigation-related neural activation in functional magnetic resonance imaging. To further illuminate the functionality of CRY2 in human navigation, we examined the correlation between CRY2 expression and various cognitive processes underlying navigation, and found high correlation of CRY2 expression with neural activity of multiple cognitive domains, particularly object and shape perception and spatial memory. Further analyses on the relation between the neural activity of human navigation and the expression maps of genes of two CRY2-related pathways, i.e., the magnetoreceptive and circadian-related functions, found a trend of correlation for the CLOCK gene, a core circadian regulator gene, suggesting that CRY2 may modulate human navigation through its role in circadian rhythm. This observation was further confirmed by a behavioral study where individuals with better circadian regularity in daily life showed better sense of direction. Taken together, our study presents the first neural evidence that links CRY2 with human navigation, possibly through the modulation of circadian rhythm.

Differential Sleep Traits Have No Causal Effect on Inflammatory Bowel Diseases: A Mendelian Randomization Study.

Background: Previous studies suggested an association of sleep disorders with inflammatory bowel disease (IBD) and indicated that using pharmacological treatments for the modulation of circadian rhythms might prevent IBD pathogenesis or aggravation, but whether the effect of sleep traits on IBD was causal is inconclusive and, therefore, prevents drug repurposing based on the previous studies. We aimed to examine the causal effect of different sleep traits on the pathogenesis of IBD. Methods: Genetic instruments for sleep traits were selected from the largest GWAS studies available in the UK Biobank (n = 449,734) and the 23andMe Research (n = 541,333). A two-sample Mendelian randomization (MR) study was conducted to examine the association of the genetic instruments with IBD (12,882 cases and 21,770 controls), ulcerative colitis (6,968 cases, 20,464 controls), and Crohn’s disease (5,956 cases and 14,927 controls). We applied the inverse-variance weighted (IVW) method to estimate causal effects, and we used the weighted median and MR-Egger method for sensitivity analyses. Results: We found that sleep duration (OR, 1.00, 95% CI 1.00–1.01), short sleep duration (OR, 1.07, 95% CI 0.41–2.83), morningness (OR, 1.05, 95% CI 0.87–1.27), daytime napping (OR, 1.64, 95% CI 0.62–4.4), frequent insomnia (OR, 1.17, 95% CI 0.8–1.72), any insomnia (OR, 1.17, 95% CI 0.69–1.97), and snoring (OR, 0.31, 95% CI 0.06–1.54) had no causal effect on IBD, and these sleep traits had no causal effect on ulcerative colitis and Crohn’s disease either. Most of the sensitivity analyses showed consistent results with those of the IVW method. Conclusion: Our MR study did not support the causal effect of sleep traits on IBD. Pharmacological modulation of circadian rhythms for the prevention of IBD pathogenesis was unwarranted.

Time-of-day dependent changes in guinea pig bladder afferent mechano-sensitivity

The voiding of urine has a clear circadian rhythm with increased voiding during active phases and decreased voiding during inactive phases. Bladder spinal afferents play a key role in the regulation of bladder storage and voiding, but it is unknown whether they exhibit themselves a potential circadian rhythm. Therefore, this study aimed to determine the mechano- and chemo- sensitivity of three major bladder afferent classes at two opposite day-night time points. Adult female guinea pigs underwent conscious voiding monitoring and bladder ex vivo single unit extracellular afferent recordings at 0300 h and 1500 h to determine day-night modulation of bladder afferent activity. All guinea pigs voided a higher amount of urine at 1500 h compared to 0300 h. This was due to an increased number of voids at 1500 h. The mechano-sensitivity of low- and high-threshold stretch-sensitive muscular-mucosal bladder afferents to mucosal stroking and stretch was significantly higher at 1500 h compared to 0300 h. Low-threshold stretch-insensitive mucosal afferent sensitivity to stroking was significantly higher at 1500 h compared to 0300 h. Further, the chemosensitivity of mucosal afferents to N-Oleoyl Dopamine (endogenous TRPV1 agonist) was also significantly increased at 1500 h compared to 0300 h. This data indicates that bladder afferents exhibit a significant time-of-day dependent variation in mechano-sensitivity which may influence urine voiding patterns. Further studies across a 24 h period are warranted to reveal potential circadian rhythm modulation of bladder afferent activity.

Melatonin supplementation in the subacute phase after ischemia alleviates postischemic sleep disturbances in rats.

BackgroundSleep disorders are highly prevalent among stroke survivors and impede stroke recovery. It is well established that melatonin has neuroprotective effects in animal models of ischemic stroke. However, as a modulator of endogenous physiological circadian rhythms, the effects of melatonin on poststroke sleep disorders remain unclear. In the present study, we investigated how melatonin delivered intraperitoneally once daily in the subacute phase after stroke onset, influencing neuronal survival, motor recovery, and sleep–wake profiles in rats.MethodsTransient ischemic stroke in male Sprague–Dawley rats was induced with 30 min occlusion of the middle cerebral artery. Melatonin or vehicle was delivered intraperitoneally once daily in the subacute phase, from 2 to 7 days after stroke. Electroencephalogram and electromyogram recordings were obtained simultaneously.ResultsCompared to the effects observed in the vehicle‐treated ischemic group, after 6 daily consecutive treatment of melatonin at 10 mg/kg starting at ischemic/reperfusion day 2, the infarct volume was significantly decreased (from 39.6 to 26.2%), and the degeneration of axons in the ipsilateral striatum and the contralateral corpus callosum were significantly alleviated. Sensorimotor performances were obviously improved as evidenced by significant increases in the latency to falling off the wire and in the use of the impaired forelimb. In addition to those predictable results of reducing brain tissue damage and mitigating behavioral deficits, repeated melatonin treatment during the subacute phase of stroke also alleviated sleep fragmentation through reducing sleep‐wake stage transitions and stage bouts, together with increasing stage durations. Furthermore, daily administration of melatonin at 9 a.m. significantly increased the nonrapid eye movement sleep delta power during both the light and dark periods and decreased the degree of reduction of the circadian index.ConclusionsMelatonin promptly reversed ischemia‐induced sleep disturbances. The neuroprotective effects of melatonin on ischemic injury may be partially associated with its role in sleep modulation.

Circadian Rhythm Modulation of Microbes During Health and Infection.

Circadian rhythms, referring to 24-h daily oscillations in biological and physiological processes, can significantly regulate host immunity to pathogens, as well as commensals, resulting in altered susceptibility to disease development. Furthermore, vaccination responses to microbes have also shown time-of-day-dependent changes in the magnitude of protective immune responses elicited in the host. Thus, understanding host circadian rhythm effects on both gut bacteria and viruses during infection is important to minimize adverse effects on health and identify optimal times for therapeutic administration to maximize therapeutic success. In this review, we summarize the circadian modulations of gut bacteria, viruses and their interactions, both in health and during infection. We also discuss the importance of chronotherapy (i.e., time-specific therapy) as a plausible therapeutic administration strategy to enhance beneficial therapeutic responses.

Cryo-EM structure of the human MT1-Gi signaling complex.

Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2 elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here we report a cryo-EM structure of the human MT1-Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gs complexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.

Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes.

Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT1 and MT2. The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[125I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive Gi/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the β-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Atomoxetine and circadian gene expression in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder

Atomoxetine (ATO) is a second line medication for attention-deficit hyperactivity disorder (ADHD). We proposed that part of the therapeutic profile of ATO may be through circadian rhythm modulation. Thus, the aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after ATO exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with a diagnosis of ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different ATO concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. No statistical significant effect of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, sleep WASO and total number of wake bouts was observed. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. ATO induced the rhythmicity of Clock in the ADHD group. This effect, however, was not observed in HDF cultures of healthy controls. Bmal1 and Per2 expression showed a significant ZT × group interaction via mixed ANOVA. Strong positive correlations for chronotype and circadian genes were observed for Bmal1, Cry1 and Per3 among the study participants. Statistical significant different Clock, Bmal1 and Per3 expressions were observed in HDFs exposed to ATO collected from ADHD participants exhibiting neutral and moderate evening preference, as well as healthy participants with morning preferences. The results of the present study illustrate that ATO impacts on circadian function, particularly on Clock, Bmal1 and Per2 gene expression.

Withanolide Derivative 2,3-Dihydro-3β-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3β-methoxy withaferin-A (3βmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3βmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3βmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 μM and that 3βmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 μM). We propose that 3βmWi-A is a novel modulator of circadian rhythms.

Reversible modulation of circadian time with chronophotopharmacology

The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.