Chronic Corticosterone Disrupts The Circadian Rhythm of CRH Expression and m6A RNA Methylation In The Chicken Hypothalamus

Background: Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. Results: Chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3’UTR of CRH mRNA indicates that higher m6A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). Conclusions: Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

The Effects of Spatial and Temporal Resolution of Gridded Meteorological Forcing on Watershed Hydrological Responses

Meteorological forcing plays a critical role in accurately simulating the watershed hydrological cycle. With the advancement of high-performance computing and the development of integrated watershed models, simulating the watershed hydrological cycle at high temporal (hourly to daily) and spatial resolution (10s of meters) has become efficient and computationally affordable. These hyperresolution watershed models require high resolution of meteorological forcing as model input to ensure the fidelity and accuracy of simulated responses. In this study, we utilized the Advanced Terrestrial Simulator (ATS), an integrated watershed model, to simulate surface and subsurface flow and land surface processes using unstructured meshes at the Coal Creek Watershed near Crested Butte (Colorado). We compared simulated watershed hydrologic responses including streamflow, and distributed variables such as evapotranspiration, snow water equivalent (SWE), and groundwater table drivenby three publicly available, gridded meteorological forcing (GMF) – Daily Surface Weather and Climatological Summaries (Daymet), Parameter-elevation Regressions on Independent Slopes Model (PRISM), and North American Land Data Assimilation System (NLDAS). By comparing various spatial resolutions (ranging from 400 m to 4 km) of PRISM, the simulated streamflow only becomes marginally worse when spatial resolution of meteorological forcing is coarsened to 4 km (or 30 % of the watershed area). However, the 4 km resolution has much worse performance than finer resolution in spatially distributedvariables such as SWE. By comparing models forced by different temporal resolutions of NLDAS (hourly to daily), GMF in sub-daily resolution preserves the dynamic watershed responses (e.g., diurnal fluctuation of streamflow) that are absent in results forced by daily resolution. Conversely, the simulated streamflow shows better performance using daily resolution compared to that using sub-daily resolution. Our findings suggest that the choice of GMF and its spatiotemporal resolution depends on the quantity of interest and its spatial and temporal scale, which may have important implications on model calibration and watershed management decisions.

Circadian dependence of the acute immune response to myocardial infarction

Aim Circadian rhythms control many physiological processes, including numerous aspects of the immune system. Whether these immunological oscillations play a role in the pathophysiology after ischemic injury, such as myocardial infarction (MI), remains unclear. In this study, we aimed to characterize circadian rhythms during the acute inflammatory responses after MI. Methods and results Balb/c mice were operated at Zeitgeber Time (ZT) 2, 8, 14 and 20. Three hours after MI, animals were terminated and blood and hearts were collected to assess immunological status and damage. We observed diurnal fluctuation in leukocyte numbers in the blood, peaking during the rest-phase (ZT2 and 8) of the circadian cycle. Interestingly, the homing and infiltration of neutrophils to the injured myocardium was more pronounced during the active-phase of the mice (ZT14 and 20), at which time higher levels of Troponin-T were measured in the serum. Higher neutrophil extravasation during the active phase (especially the sleep-to-wake transition, ZT14) was also correlated to greater chemokine release in the blood and adhesion molecule expression in the heart. Conclusion The occurrence of a myocardial infarction during the early-morning hours leads to greater myocardial damage in patients. In the present study, we showed that the neutrophil response in the first hours after MI is stronger during the sleep-to-wake transition, thereby potentially playing a role in the worse clinical outcome observed during this time period. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research received funding from the EU's H2020 research and innovation programme under Marie S. Curie cofund RESCUE grant agreement No 801540.

Chronic Corticosterone Disrupts the Circadian Rhythm of CRH Expression and M6a RNA Methylation in the Chicken Hypothalamus

Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. Here, we show that chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3’UTR of CRH mRNA indicates that higher m6A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

Faecal Cortisol Metabolites as an Indicator of Adrenocortical Activity in Farmed Blue Foxes

Welfare studies of blue foxes would benefit from a measurement of faecal cortisol metabolites (FCMs) as a non-invasive, physiological stress parameter reflecting hypothalamus–pituitary–adrenal (HPA) axis activity. Before implementation, a species-specific validation of such a method is required. Therefore, we conducted a physiological validation of an enzyme immunoassay (EIA) to measure FCMs in blue foxes. Twenty individuals (nine males and eleven females) were injected with synthetic adrenocorticotrophic hormone (ACTH) and faecal samples were collected every third h for two days. The FCM baseline levels were assessed based on the first sampling day (control period, 144 samples), followed by the ACTH injection and the second day of sampling (treatment period, 122 samples). FCMs were analysed with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA. We compared the estimated mean FCM concentrations of the treatment samples to the baseline average. All samples for the two periods were collected at the same time of the day, which enabled to test the data also with an hourly pairwise comparison. With the two statistical approaches, we tested whether a possible diurnal fluctuation in the FCM concentrations affected the interpretation of the results. Compared to the baseline levels, both approaches showed 2.4–3.2 times higher concentrations on time points sampled 8–14 h after the ACTH injection (p < 0.05). The estimated FCM concentrations also fluctuated slightly within the control period (p < 0.01). Inter-individual variations in FCM levels were marked, which highlights the importance of having a sufficient number of animals in experiments utilising FCMs. The sampling intervals of 3 h enabled forming of informative FCM curves. Taken together, this study proves that FCM analysis with a 5α-pregnane-3ß,11ß,21-triol-20-one EIA is a valid measurement of adrenocortical activity in the farmed blue foxes. Therefore, it can be utilised as a non-invasive stress indicator in future animal welfare studies of the species.

Recognizing Atypical Dopa-Responsive Dystonia and Its Mimics

ABSTRACTPurpose of ReviewDopa-responsive dystonia (DRD) encompasses a group of phenotypically and genetically heterogeneous neurochemical disorders. Classic GTP cyclohydrolase 1 (GCH-1)-associated DRD consists of early-onset lower limb asymetrical dystonia, with sleep benefit, diurnal variation, and excellent and sustained response to low L-dopa doses.Recent findingsUnlike the classic phenotype, GCH-1-associated DRD may include features inconsistent with the original phenotype. We describe a GCH-1-associated late-onset DRD case with family history of parkinsonism and cervical dystonia whose response to levodopa was poor and complicated with dyskinesia, blepharospasm and severe non-motor symptoms. We use this case as a springboard to review the spectrum of atypical DRD, DRD-plus and DRD mimics.SummaryGCH-1-related dystonia may exhibit wide intrafamilial phenotypic variability, no diurnal fluctuation, poor response to L-dopa, and such complications as dyskinesia, epilepsy, sleep disorders, autonomic dysfunction, oculogyric crisis, myoclonus, or tics. More recently, rare GCH-1 variants have been found to be associated with Parkinson's disease. Clinicians should be aware of atypical DRD, DRD-plus and DRD mimics.

Identification of TH Variants in Chinese Dopa-Responsive Dystonia Patients and Long-Term Outcomes

Background: Dopa-responsive dystonia (DRD) is a movement disorder that is highly clinically and genetically heterogeneous. Our study summarizes clinical characteristics and long-term outcomes in patients with dopa-responsive dystonia with the aim of obtaining further knowledge on this disorder.Methods: Patients who met DRD genetic diagnostic criteria through whole-exome sequencing and took levodopa for over 3 years were included in our study. Detailed information was collected on these patients, including family history, age at onset, age and dosage at starting levodopa, current medication and dosage, levodopa duration, diurnal fluctuation, and other clinical features. The Burke–Fahn–Marsden Dystonia Rating Scale-Motor (BFMDRS-M) score was used to evaluate patients' dystonia and variation after levodopa. According to the long-term outcomes, patients were further graded as good (dystonia improved by more than 50% after levodopa, and no further motor symptoms appeared) and poor (dystonia improved by &lt;50% after levodopa, or new motor symptoms appeared).Results: A total of 20 DRD patients were included (11 with GCH1 variants, 9 with TH variants). During long-term levodopa treatment, three patients with TH variants (3/20, 15%) developed motor symptoms, including body jerks and paroxysmal symptoms, and responded well to increasing levodopa doses. The patient with homozygous mutation c.1481C&gt;T/p. Thr494Met harbored more serious symptoms and poor response to levodopa and showed decreased cardiac uptake in MIBG.Conclusions: Most DRD patients showed satisfactory treatment outcomes after long-term levodopa, whereas few patients with TH variants presented motor symptoms, which is considered to be related to dopamine insufficiency. For patients with motor symptoms after long-term levodopa, increasing the dose slowly might be helpful to relieve symptoms.

Case Report: Guitarist’s cramp as the initial manifestation of dopa-responsive dystonia with a novel heterozygous GCH1 mutation

Dopa-responsive dystonia (DRD), also known as Segawa syndrome, is a phenotypically and genetically heterogeneous group of neurological disorders that typically presents as early-onset lower limb dystonia with diurnal fluctuation, and exhibits a marked, persistent response to levodopa. Heterozygous loss-of-function mutations in the guanosine triphosphate cyclohydrolase 1 (GCH1) are the most common cause of DRD. In addition to the classic form of the disease, there have been a number of studies addressing atypical clinical features of GCH1 related DRD with variable age of onset. This report describes a 37-year-old Japanese male patient with a 10-year history of focal upper limb dystonia that initially emerged as task-specific, guitarist’s cramp. The dystonic symptoms responded very well to levodopa treatment, and genetic analysis identified a novel heterozygous mutation in the C-terminal catalytic domain of GCH1. Insufficient recognition of this treatable condition often leads to misdiagnosis, which causes delays in the patient receiving adequate dopamine replenishing therapy. A diagnostic trial with levodopa should be considered in all patients with relatively young-onset dystonia, whether they have classic features of DRD or not.

Utility of Urinary Ratio of Cortisol to Aldosterone as Inflammatory and Metabolic Parameters

Corticosteroids are important bioactive substances in the body that regulate inflammation, metabolism, immunity, and circulatory dynamics. Cortisol and aldosterone are two major naturally occurring steroids, which synthesized in the adrenal fasciculata and in the adrenal glomerulosa under the regulation of adrenocorticotropin (ACTH) and renin-angiotensin system (RAS), respectively. The conventional strategy for evaluating adrenocortical function has been to measure random serum cortisol or 24-hour stored levels of urine free cortisol (UFC). UFC is a more appropriate marker to avoid the effects of diurnal fluctuation of cortisol, serum cortisol-binding proteins, and cortisol clearance in the kidney. Thus, measurement of UFC has been a reliable test for diagnosis of Cushing’s disease or adrenal insufficiency. However, since the normal range of UFC varies widely, it is often difficult to evaluate whether the UFC level is optimal or not for each patient. That is because UFC is greatly affected by the amount of fluid intake or urine volume, and an immunoassay for UFC, which is the usual method for measuring UFC, is susceptible to interference from other steroid metabolites and synthetic glucocorticoids. To explore an alternative indicator, we tried to standardize the levels of UFC by the levels of urinary aldosterone concentration (UAC) in the same urinary sample. Medical records of all 246 patients in whom daily excretions of UFC and UAC had been measured between 2015 and 2018 at our department were reviewed. 142 patients (including 93 females) were included after exclusion of 104 patients because of corticosteroid replacement therapy. UFC/UAC ratio showed significant positive and negative correlations with the levels of serum cortisol (R=0.287) and aldosterone (R=-0.762), respectively. UFC/UAC ratio increased with aging in female patients, while the ratio was not altered by the levels of BMI in either gender. Markers for metabolic and inflammatory status including hemoglobin A1c (R=0.327), albumin (R=-0.331), chorine esterase (R=-0.248), C-reactive protein (R=0.317), ferritin (R=0.473), and D-dimer (R=0.569) showed correlations to the ratio of UFC/UAC that were more significant than the correlations to the serum level of cortisol or UFC alone. Of note, the UFC/UAC ratio was shown as an indicator for risks of diabetes (AUC: 0.765), hypoalbuminemia (0.839), hyper-CRPemia (0.748), and thrombophilia (0.824), in which the cut-off levels of UFC/UAC ratios were found to be around 12. These results indicate that the UFC/UAC ratio is a suitable variable for the detection of metabolic and inflammatory complications related to adrenocortical dysfunction.

The Arabidopsis JMJ29 Protein Controls Circadian Oscillation through Diurnal Histone Demethylation at the CCA1 and PRR9 Loci

The circadian clock matches various biological processes to diurnal environmental cycles, such as light and temperature. Accumulating evidence shows that chromatin modification is crucial for robust circadian oscillation in plants, although chromatin modifiers involved in regulating core clock gene expression have been limitedly investigated. Here, we report that the Jumonji C domain-containing histone demethylase JMJ29, which belongs to the JHDM2/KDM3 group, shapes rhythmic changes in H3K4me3 histone marks at core clock loci in Arabidopsis. The evening-expressed JMJ29 protein interacts with the Evening Complex (EC) component EARLY FLOWERING 3 (ELF3). The EC recruits JMJ29 to the CCA1 and PRR9 promoters to catalyze the H3K4me3 demethylation at the cognate loci, maintaining a low-level expression during the evening time. Together, our findings demonstrate that interaction of circadian components with chromatin-related proteins underlies diurnal fluctuation of chromatin structures to maintain circadian waveforms in plants.