Stem Cell Secretome Modulated by Arsenicum album 30C Ameliorates Lipopolysaccharide-induced Cytokine Storm in Blood Mononuclear Cells in vitro.

BACKGROUND: The therapeutic effectiveness of mesenchymal stem cells (MSCs) and their secretome can be enhanced by means of physical, chemical and biological preconditioning. Arsenicum album 30C (AA30) has been one of the leading homeopathic medicines used in prophylaxis against SARS-CoV-2 infection. AIMS: This study aimed to investigate whether AA30 preconditioning could influence the growth factors and cytokine profile of the human dental pulp-derived MSC (DPD-MSC) secretome. Also, to test the efficacy of the AA30-preconditioned DPD-MSC secretome in ameliorating the lipopolysaccharide (LPS)-induced cytokine storm in human peripheral blood mononuclear cells (PBMCs) as an in-vitro cellular model. METHODS: The cytotoxicity of AA30 was assessed in DPD-MSCs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Growth factors and cytokine levels in the AA30-preconditioned DPD-MSC secretome were analysed by fluorescence-activated cell sorting (FACS) analysis. The angiogenic potential of the AA30-preconditioned DPD-MSC secretome was assessed by chick yolk-sac membrane (YSM) assay. Culture medium with 0.001% ethanol was used as vehicle control. The efficacy of the AA30-preconditioned DPD-MSC secretome in ameliorating the cytokine storm was assessed in LPS pre-treated PBMCs. The mRNA and protein expression of inflammatory markers such as IL-1ß, IL-6 and IL-10 were analysed by using RT-PCR and FACS analysis respectively. RESULTS: AA30 did not exhibit cytotoxicity in the concentration range of 1% to 50%. Furthermore, the AA30-preconditioned DPD-MSC secretome exhibited a significant increase in the levels of angiogenic factors, such as human angiopoietin-2, EPO and PDGF-AA, and decreased levels of cytokines, such as TNF-α, CXCL-8 and IL-6. The AA30-preconditioned DPD-MSC secretome showed augmented angiogenesis compared to vehicle controls. The DPD-MSC secretome ameliorated LPS-induced mRNA and protein expression of IL-1ß, IL-6 and IL-10 in PBMCs. CONCLUSION: The AA30-preconditioned DPD-MSC secretome augmented angiogenesis and ameliorated the LPS-induced cytokine storm in human PBMCs in vitro. Our data demonstrate that AA30 preconditioning enhances the therapeutic potency of MSCs and their secretome.

Severity and mortality associated with COVID-19 among children hospitalised in tertiary care centres in India: a cohort study.

Background: It is critical to identify high-risk groups among children with COVID-19 from low-income and middle-income countries (LMICs) to facilitate the optimum use of health system resources. The study aims to describe the severity and mortality of different clinical phenotypes of COVID-19 in a large cohort of children admitted to tertiary care hospitals in India. Methods: Children aged 0-19 years with evidence of SARS-CoV-2 infection (real time polymerase chain reaction or rapid antigen test positive) or exposure (anti-SARS-CoV-2 antibody, or history of contact with SARS-CoV-2) were enrolled in the study, between January 2021 and March 2022 across five tertiary hospitals in India. All study participants enrolled prospectively and retrospectively were followed up for three months after discharge. COVID-19 was classified into severe (Multisystem Inflammatory Syndrome in Children (MIS-C), severe acute COVID-19, 'unclassified') or non-severe disease. The mortality rates were estimated in different phenotypes. Findings: Among 2468 eligible children enrolled, 2148 were hospitalised. Signs of illness were present in 1688 (79%) children with 1090 (65%) having severe disease. High mortality was reported in MIS-C (18.6%), severe acute COVID-19 (13.3%) and the unclassified severe COVID-19 disease (12.3%). Mortality remained high (17.5%) when modified MIS-C criteria was used. Non-severe COVID-19 disease had 14.1% mortality when associated with comorbidity. Interpretation: Our findings have important public health implications for low resource settings. The high mortality underscores the need for better preparedness for timely diagnosis and management of COVID-19. Children with associated comorbidity or coinfections are a vulnerable group and need special attention. MIS-C requires context specific diagnostic criteria for low resource settings. It is important to evaluate the clinical, epidemiological and health system-related risk factors associated with severe COVID-19 and mortality in children from LMICs. Funding: Department of Biotechnology, Govt of India and Department of Maternal, Child and Adolescent Health and Aging, WHO, Geneva, Switzerland.

A Survey to Assess the Impact of COVID-19 Pandemic on Lifestyle in an Adult Age Group.

Context: A healthy lifestyle has been the need of the hour during the COVID 19 pandemic. Analyzing the current lifestyle patterns of many individuals can be the basis for finding solutions toward building a healthy future for India. Objectives: The study intended to evaluate the current lifestyles of adults in an urban setting in the midst of a pandemic and to examine the diseases that people could face with respect to their current lifestyles. Design: The research team performed a cross-sectional study. Setting: The survey was conducted in an urban setting in the Pimpri Chinchwad area of Pune, India. Participants: Participants were 500 men and women between the ages of 18 and 25. Outcome Measures: The research team created a survey with 13 multiple-choice questions. Results: The pandemic has taken a toll on people's mental and physical health. Social distancing and staying indoors for long periods are factors that have affected people's mental health. Conclusions: Efforts need to be made by individuals to focus not only on their physical health but also on their mental health.

Breast Abscess Healing with Homoeopathy: A Case Report.

Context: Breast abscess is the most common complication of acute bacterial mastitis usually referred to as pyogenic mastitis. It is usually encountered during lactation due to an infection with Staphylococcus aureus and streptococcal bacteria. These bacteria produce a severe inflammatory reaction leading to pus formation which is mainly treated by ultrasound-guided drainage or fine needle aspirations. We find that in this condition homoeopathic treatment can play an important role as it avoids such surgical procedures and helps in healing in a most gentle and rapid way. We report a successful single case study that opens up opportunities to take up more such cases to strengthen the results of this report. Case summary: A 23-year-old lactating mother came with complaints of pain and swelling with a collection of pus in the breast region and decreased breast milk secretion. Individualised homoeopathic medicine Silicea 200C was given to hasten suppuration and Belladonna 200C to treat inflammation.Thus, this case shows us how only few doses of medicine may be quickly helpful in managing a case of breast abscess without the need of any surgical procedures.

Conventional Homeopathic Medicine and Its Relevance to Modern Medicine.

Context: Homeopathic medicine can be explained as a symptoms-based method of treatment, and it can act as an alternative treatment strategy against allopathy by focusing on the symptoms of illness, as opposed to causative agents as allopathic medicine does. Also, homeopathic medicines are extracted from nature rather than being chemically synthesized as western drugs are. Objective: The review intended to briefly describe the concept of homeopathic medicine, its emergence from a historical point of view, and its broader healing properties, providing examples of key homeopathic drugs and comparing them to modern medicines. Design: The research team performed a narrative review by searching databases like Pubmed, Google Scholar, and other national search engines. The search used the keywords homeopathic medicine, alternate medicine, materia medica, allium cepa, Zingiber officinale, penicillium, Agaricus muscaria, Botulinum toxin. Setting: Dr. D.Y. Patil Homoeopathic Medical College and Research Centre, Dr. D.Y. Patil Vidyapeeth (Deemed to be University), Pimpri, Pune. Results: This review highlights the rich sources homoeopathic drugs and their corelation with modern medicine. The current review focuses on the significance of the Homeopathic Materia Medica and on notable remedies in homeopathy that align with allopathy in addressing different pathological conditions, including treatments that the two types of medicine have in in common and that are effective in homeopathy. Conclusions: Many studies are being conducted to prove the mechanism of action of homoeopathic medicines. Droplet Evaporating Method (DEM), Raman, UltraViolet-Visible (UV-VIS) spectroscopy and Transmission Electron Microscopy (TEM) are commonly used methods to characterize homeopathic medicines at ultra-low concentration and many such studies will surely indicate how homoeopathic medicines act. Such research results may subsequently lead to the betterment of treatment procedures and the integration of homeopathic principles into mainstream medical practices.

Advance Teaching of Fundamentals of Homoeopathy (ATFH): redefining competency based approach

The curriculumin academic medicine is defined by writing effective Learning objectives (LO). LO iselaborated based on perceptions of unbiased written aspects,of course, the rationale in statements is explained and tested through the completion of educational activity. These are the foundations for defining the outcomes in building up strong educational policieswhich are instructionalaligned through predefined effective curriculum courseswith added mapped outcomes. This letter provides the ongoing aspect of the development of Homoeopathic education in India regulated by the National Commission for Homoeopathy for the subject course of Advance Teaching of Fundamentals of Homoeopathy (ATFH). The essential components for the ATFHsubject course with LO and outcome assessment is been discussed and would provide a new arena of academic research in building up rationale in the programed [Doctor of Homoeopathy(MD,(Hom).

Polycystic Ovarian Syndrome Treated with Individualized Homeopathy: A Case Report.

Introduction: Polycystic ovarian syndrome (PCOS) is a polygenic, multifactorial, syndromic disorder with reproductive, endocrine, and metabolic dysfunction seen in reproductive aged women (12-45 years). The exact cause is not known may involve increased luteinizing hormone, increased insulin levels, and a defect in androgen synthesis. The symptoms include anovulation, irregular menses, and hyperandrogenism. It is clinically manifested by hirsutism, acne, and androgenic alopecia. Health care practitioners continue to seek a cure for PCOS as it is increasing in frequency and is one of the major causes of anovulatory infertility. Methods: The case was recorded in the gynaecological department at the Homoeopathic Medical College and Research Centre. An 18- year-old female patient with PCOS was treated with individualised homeopathy (iHOM) medicine between 26th September 2019 and 26th November 2020. During the follow-up visits, treatment outcomes were assessed. To assess whether the changes were due to homoeopathic medicine, an assessment using the modified Naranjo criteria was performed. Results: Over an observational period of 1 year, beneficial result from iHOM medicine was seen. This treatment method can be used by the physicians in the treatment of PCOS as a complementary health practice. Conclusion: Considering the multi-factorial aetiology of PCOS, iHOM medicine with lifestyle modification is helpful in treating PCOS.

COVID-19 vaccination hesitancy in India: State of the nation and priorities for research.

BACKGROUND: Few COVID-19 vaccines were anticipated in India in early 2021. However, little was known about COVID-19 vaccination acceptance among the public. We conducted a nationwide study to understand the public's perception about COVID-19 vaccines in December 2020. METHOD: An online survey was deployed using a multi-item validated questionnaire via social media websites and networking platforms for adults in India. We asked participants about vaccination willingness, concerns about vaccination, and their sociodemographic characteristics. RESULTS: Nationwide, 1638 participants from 27 states/union territories took the survey where the majority of the participant were males (55%), 18-30 years old (52%), urban dwellers (69%), college-educated (81%), without a history of COVID-19 infection (92%). More than a fifth were either unaware of the vaccines (20.63%) or were not sure if they will get the vaccine (27%), and 10% refused to obtain the vaccine. Almost 70% of the population had concerns regarding the vaccines. Statistically significant differences (p<0.01) in awareness about vaccine and acceptability were observed based on age, educational qualifications, and employment status. CONCLUSION: While the majority of Indians would accept the vaccine, given the large population of India, even a small proportion of hesitant individuals would translate to millions of unvaccinated individuals. Strategic measures and policy decisions to enhance the rate of COVID-19 vaccination should be continuously planned and implemented in India.

Longitudinal Serology of SARS-CoV-2-Infected Individuals in India: A Prospective Cohort Study.

Clinical and epidemiological characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now widely available, but there are few data regarding longitudinal serology in large cohorts, particularly those from low-income and middle-income countries. We established an ongoing prospective cohort of 3,840 SARS-CoV-2-positive individuals according to RT-PCR in the Delhi-National Capital Region of India to document clinical and immunological characteristics during illness and convalescence. The immunoglobulin G (IgG) responses to the receptor binding domain (RBD) and nucleocapsid were assessed at 0 to 7 days, 10 to 28 days, and 6 to 10 weeks after infection. The clinical predictors of seroconversion were identified by multivariable regression analysis. The seroconversion rates during the postinfection windows of 0 to 7 days, 10 to 28 days, and 6 to 10 weeks were 46%, 84.7%, and 85.3%, respectively (N = 743). The proportion with a serological response increased with the severity of coronavirus disease 2019 (COVID-19). All participants with severe disease, 89.6% with mild to moderate infection, and 77.3% of asymptomatic participants had IgG antibodies to the RBD antigen. The threshold values for the nasopharyngeal viral RNA RT-PCR of a subset of asymptomatic and symptomatic seroconverters were comparable (P = 0.48) to those of nonseroconverters (P = 0.16) (N = 169). This is the first report of longitudinal humoral immune responses to SARS-CoV-2 over a period of 10 weeks in South Asia. The low seropositivity of asymptomatic participants and differences between assays highlight the importance of contextualizing the understanding of population serosurveys.

Indirect Estimation of Vertical Ground Reaction Force from a Body-Mounted INS/GPS Using Machine Learning.

Vertical ground reaction force (vGRF) can be measured by force plates or instrumented treadmills, but their application is limited to indoor environments. Insoles remove this restriction but suffer from low durability (several hundred hours). Therefore, interest in the indirect estimation of vGRF using inertial measurement units and machine learning techniques has increased. This paper presents a methodology for indirectly estimating vGRF and other features used in gait analysis from measurements of a wearable GPS-aided inertial navigation system (INS/GPS) device. A set of 27 features was extracted from the INS/GPS data. Feature analysis showed that six of these features suffice to provide precise estimates of 11 different gait parameters. Bagged ensembles of regression trees were then trained and used for predicting gait parameters for a dataset from the test subject from whom the training data were collected and for a dataset from a subject for whom no training data were available. The prediction accuracies for the latter were significantly worse than for the first subject but still sufficiently good. K-nearest neighbor (KNN) and long short-term memory (LSTM) neural networks were then used for predicting vGRF and ground contact times. The KNN yielded a lower normalized root mean square error than the neural network for vGRF predictions but cannot detect new patterns in force curves.

An Automatic Aggregator of Power Flexibility in Smart Buildings Using Software Based Orchestration.

This paper presents a software-based modular and hierarchical building energy management system (BEMS) to control the power consumption in sensor-equipped buildings. In addition, the need of this type of solution is also highlighted by presenting the worldwide trends of thermal energy end use in buildings and peak power problems. Buildings are critical component of smart grid environments and bottom-up BEMS solutions are need of the hour to optimize the consumption and to provide consumption side flexibility. This system is able to aggregate the controls of the all-controllable resources in building to realize its flexible power capacity. This system provides a solution for consumer to aggregate the controls of 'behind-the-meter' small loads in short response and provide 'deep' demand-side flexibility. This system is capable of discovery, status check, control and management of networked loads. The main novelty of this solution is that it can handle the heterogeneity of the installed hardware system along with time bound changes in the load device network and its scalability; resulting in low maintenance requirements after deployment. The control execution latency (including data logging) of this BEMS system for an external control signal is less than one second per connected load. In addition, the system is capable of overriding the external control signal in order to maintain consumer coziness within the comfort temperature thresholds. This system provides a way forward in future for the estimation of the energy stored in the buildings in the form of heat/temperature and use buildings as temporary batteries when electricity supply is constrained or abundant.

The Bdnf and Npas4 genes are targets of HDAC3-mediated transcriptional repression.

BACKGROUND: Histone deacetylase-3 (HDAC3) promotes neurodegeneration in various cell culture and in vivo models of neurodegeneration but the mechanism by which HDAC3 exerts neurotoxicity is not known. HDAC3 is known to be a transcriptional co-repressor. The goal of this study was to identify transcriptional targets of HDAC3 in an attempt to understand how it promotes neurodegeneration. RESULTS: We used chromatin immunoprecipitation analysis coupled with deep sequencing (ChIP-Seq) to identify potential targets of HDAC3 in cerebellar granule neurons. One of the genes identified was the activity-dependent and neuroprotective transcription factor, Neuronal PAS Domain Protein 4 (Npas4). We confirmed using ChIP that in healthy neurons HDAC3 associates weakly with the Npas4 promoter, however, this association is robustly increased in neurons primed to die. We find that HDAC3 also associates differentially with the brain-derived neurotrophic factor (Bdnf) gene promoter, with higher association in dying neurons. In contrast, association of HDAC3 with the promoters of other neuroprotective genes, including those encoding c-Fos, FoxP1 and Stat3, was barely detectable in both healthy and dying neurons. Overexpression of HDAC3 leads to a suppression of Npas4 and Bdnf expression in cortical neurons and treatment with RGFP966, a chemical inhibitor of HDAC3, resulted in upregulation of their expression. Expression of HDAC3 also repressed Npas4 and Bdnf promoter activity. CONCLUSION: Our results suggest that Bdnf and Npas4 are transcriptional targets of Hdac3-mediated repression. HDAC3 inhibitors have been shown to protect against behavioral deficits and neuronal loss in mouse models of neurodegeneration and it is possible that these inhibitors work by upregulating neuroprotective genes like Bdnf and Npas4.

Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.

The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.

Continuous Analysis of Running Mechanics by Means of an Integrated INS/GPS Device.

This paper describes a single body-mounted sensor that integrates accelerometers, gyroscopes, compasses, barometers, a GPS receiver, and a methodology to process the data for biomechanical studies. The sensor and its data processing system can accurately compute the speed, acceleration, angular velocity, and angular orientation at an output rate of 400 Hz and has the ability to collect large volumes of ecologically-valid data. The system also segments steps and computes metrics for each step. We analyzed the sensitivity of these metrics to changing the start time of the gait cycle. Along with traditional metrics, such as cadence, speed, step length, and vertical oscillation, this system estimates ground contact time and ground reaction forces using machine learning techniques. This equipment is less expensive and cumbersome than the currently used alternatives: Optical tracking systems, in-shoe pressure measurement systems, and force plates. Another advantage, compared to existing methods, is that natural movement is not impeded at the expense of measurement accuracy. The proposed technology could be applied to different sports and activities, including walking, running, motion disorder diagnosis, and geriatric studies. In this paper, we present the results of tests in which the system performed real-time estimation of some parameters of walking and running which are relevant to biomechanical research. Contact time and ground reaction forces computed by the neural network were found to be as accurate as those obtained by an in-shoe pressure measurement system.

Estradiol (E2)- and tamoxifen (Tmx)-bound ER-alpha (ERα) interact differentially with histone deacetylases 1 and 3 (HDACs 1 and 3).

Although ERα activation properties have been intensively studied, this is not the case for their repressive properties. In this report, the ERα ligand binding domain (LBD) is shown to interact both with a deacetylase function and with HDAC1 and HDAC3. Ligands do not affect binding to the deacetylase activity or to HDAC1. In distinction, E2 reduced LBD binding to HDAC3, whereas Tmx had no effect. Knock-down of either HDAC1 or 3 led to increased transcriptional activity by both HDACs, presumably by decreased repression. In distinction, only HDAC3 knock-down led to increased activity in the presence of Tmx. In summary, ERα differentially interacts with HDACs 1 and 3 to regulate transcriptional activity.

ß2-spectrin depletion impairs DNA damage repair.

ß2-Spectrin (ß2SP/SPTBN1, gene SPTBN1) is a key TGF-ß/SMAD3/4 adaptor and transcriptional cofactor that regulates TGF-ß signaling and can contribute to liver cancer development. Here we report that cells deficient in ß2-Spectrin (ß2SP) are moderately sensitive to ionizing radiation (IR) and extremely sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with IR or ICL agents (formaldehyde, cisplatin, camptothecin, mitomycin), ß2SP deficient cells displayed a higher frequency of cells with delayed γ-H2AX removal and a higher frequency of residual chromosome aberrations. Following hydroxyurea (HU)-induced replication stress, ß2SP-deficient cells displayed delayed disappearance of γ-H2AX foci along with defective repair factor recruitment (MRE11, CtIP, RAD51, RPA, and FANCD2) as well as defective restart of stalled replication forks. Repair factor recruitment is a prerequisite for initiation of DNA damage repair by the homologous recombination (HR) pathway, which was also defective in ß2SP deficient cells. We propose that ß2SP is required for maintaining genomic stability following replication fork stalling, whether induced by either ICL damage or replicative stress, by facilitating fork regression as well as DNA damage repair by homologous recombination.

Transcriptome profiling of expression changes during neuronal death by RNA-Seq.

The molecular mechanisms underlying neuronal death are poorly understood. One of the most widely used models to study neuronal death are cultured cerebellar granule neurons (CGNs) which undergo apoptosis when switched from a medium containing depolarizing levels of potassium (HK) to a medium with low non-depolarizing levels of potassium (LK). Previously, other labs have used DNA microarray analysis to characterize gene expression changes in LK-treated CGNs. However, microarray analysis is only capable of measuring the status of known transcripts, and expression of low-abundance mRNAs is often not detected by the hybridization-based approach. We have used RNA-sequencing to conduct a more detailed and comprehensive analysis of gene expression changes in CGNs induced to die by LK treatment. RNA-seq investigates the status of both known transcripts as well as exploring new ones and is substantially more sensitive than the microarray approach. We have found that the expression of 4334 genes is significantly altered in LK-treated CGNs with 2199 being up-regulated while 2135 are down-regulated. Genes functioning in cell death and survival regulation, cell growth and proliferation and molecular transport were most affected by LK treatment. Further, a large number of genes involved in nervous system development and function were also deregulated. Analysis of signaling pathways that were affected in LK-induced death included but were not limited to mitochondrial dysfunction and oxidative phosphorylation, consistent with a number of studies showing perturbations of these pathways in neurodegenerative disorders. Thus, our study identifies a large number of new genes that are affected during the process of neuronal death. While a majority of these changes may reflect consequences of the induction of neuronal death, many of the genes that we have identified are likely to be critical and potentially novel mediators of neuronal death, including death associated with neurodegenerative disease.

Histone deacetylase 3 is necessary for proper brain development.

The functional role of histone deacetylase 3 (HDAC3) in the developing brain has yet to be elucidated. We show that mice lacking HDAC3 in neurons and glia of the central nervous system, Nes-Cre/HDAC3 conditional KO mice, show major abnormalities in the cytoarchitecture of the neocortex and cerebellum and die within 24 h of birth. Later-born neurons do not localize properly in the cortex. A similar mislocalization is observed with cerebellar Purkinje neurons. Although the proportion of astrocytes is higher than normal, the numbers of oligodendrocytes are reduced. In contrast, conditional knockout of HDAC3 in neurons of the forebrain and certain other brain regions, using Thy1-Cre and calcium/calmodulin dependent protein kinase II α-Cre for ablation, produces no overt abnormalities in the organization of cells within the cortex or of cerebellar Purkinje neurons at birth. However, both lines of conditional knockout mice suffer from progressive hind limb paralysis and ataxia and die around 6 weeks after birth. The mice display an increase in overall numbers of cells, higher numbers of astrocytes, and Purkinje neuron degeneration. Taken together, our results demonstrate that HDAC3 plays an essential role in regulating brain development, with effects on both neurons and glia in different brain regions.

Dexamethasone induces a putative repressor complex and chromatin modifications in the CRH promoter.

Glucocorticoids down-regulate expression of hypothalamic CRH; however, mechanisms by which they do so are not fully understood. The proximal promoter cAMP response element, negative glucocorticoid response element (nGRE), and methylated CpG islands all play a role in crh down-regulation. Dexamethasone (Dex)-repressed crh expression is associated with glucocorticoid receptor (GR) and histone deacetylase 1 (HDAC1) recruitment to the region of the crh promoter. Given that HDAC1 may be present in methylated CpG binding protein 2 (MeCP2) complexes, and that MeCP2 is known to play a role in regulating crh expression, we sought to determine whether or not HDAC1 and/or MeCP2 could interact with the GR. Dex enhanced GR interactions with both proteins. Glucocorticoid regulation of crh has also been associated with CpG methylation; thus we assessed whether GR could interact with a DNA methyltransferase (DnMT). Indeed, the GR interacted with DnMT3b, but not DnMT3a. In addition, Dex-induced occupancy of the crh promoter by HDAC1, MeCP2, and DnMT3b was associated with an increased level of promoter methylation, which appeared to be CpG site specific. Lastly, to extend previous assessment of chromatin modifications in this promoter region, the degree of histone methylation was measured. Dex increased trimethylation of histone 3-lysine 9, a marker of gene suppression; however, levels of di- and trimethylated histone 3-lysine 4, markers of gene activation, were not significantly changed. Taken together, the data suggest that Dex-mediated crh suppression involves formation of a repressor complex consisting of GR, MeCP2, and HDAC1, recruitment of DnMT3b, and associated changes in proximal promoter CpG methylation.

The ERß ligand 5α-androstane, 3ß,17ß-diol (3ß-diol) regulates hypothalamic oxytocin (Oxt) gene expression.

The endocrine component of the stress response is regulated by glucocorticoids and sex steroids. Testosterone down-regulates hypothalamic-pituitary-adrenal (HPA) axis activity; however, the mechanisms by which it does so are poorly understood. A candidate testosterone target is the oxytocin gene (Oxt), given that it too inhibits HPA activity. Within the paraventricular nucleus of the hypothalamus, oxytocinergic neurons involved in regulating the stress response do not express androgen receptors but do express estrogen receptor-ß (ERß), which binds the dihydrotestosterone metabolite 3ß,17ß-diol (3ß-diol). Testosterone regulation of the HPA axis thus appears to involve the conversion to the ERß-selective ligand 5α-androstane, 3ß-diol. To study mechanisms by which 3ß-diol could regulate Oxt expression, we used a hypothalamic neuronal cell line derived from embryonic mice that expresses Oxt constitutively and compared 3ß-diol with estradiol (E2) effects. E2 and 3ß-diol elicited a phasic response in Oxt mRNA levels. In the presence of either ligand, Oxt mRNA levels were increased for at least 60 min and returned to baseline by 2 h. ERß occupancy preceded an increase in Oxt mRNA levels in the presence of 3ß-diol but not E2. In tandem with ERß occupancy, 3ß-diol increased occupancy of the Oxt promoter by cAMP response element-binding protein and steroid receptor coactivator-1 at 30 min. At the same time, 3ß-diol led to the increased acetylation of histone H4 but not H3. Taken together, the data suggest that in the presence of 3ß-diol, ERß associates with cAMP response element-binding protein and steroid receptor coactivator-1 to form a functional complex that drives Oxt gene expression.