H3F3A K27M Mutations Drives a Repressive Transcriptome by Modulating Chromatin Accessibility, Independent of H3K27me3 in Diffuse Midline Glioma.

Background: Heterozygous histone H3.3K27M mutation is a primary oncogenic driver of Diffuse Midline Glioma (DMG). H3.3K27M inhibits the Polycomb Repressive Complex 2 (PRC2) methyltransferase complex, leading to a global reduction and redistributing of the repressive H3 lysine 27 tri-methylation. This rewiring of the epigenome is thought to promote gliomagenesis. Methods: We established novel, isogenic DMG patient-derived cell lines that have been CRISPR-Cas9 edited to H3.3 WT or H3.3K27M alone and in combination with EZH2 and EZH1 co-deletion, inactivating PRC2 methyltransferase activity of PRC2 and eliminating H3K27me3. Results: RNA-seq and ATAC-seq analysis of these cells revealed that K27M has a novel epigenetic effect that appears entirely independent of its effects on PRC2 function. While the loss of the PRC2 complex led to a systemic induction of gene expression (including HOX gene clusters) and upregulation of biological pathways, K27M led to a balanced gene deregulation but having an overall repressive effect on the biological pathways. Importantly, the genes uniquely deregulated by the K27M mutation, independent of methylation loss, are closely associated with changes in chromatin accessibility, with upregulated genes becoming more accessible. Notably, the PRC2- independent function of K27M appears necessary for tumorigenesis as xenografts of our H3.3K27M/EZH1/2 WT cells developed into tumors, while H3.3/EZH1/2 KO cells did not. Conclusion: We demonstrate that K27M mutation alters chromatin accessibility and uniquely deregulates genes, independent of K27 methylation. We further show the mutation's role in altering biological pathways and its necessity for tumor development. Key Points: We revealed genes regulated by H3.3K27M mutation and PRC2 in DMG.H3.3K27M mutation alters chromosome accessibility independent of H3K27me3.PRC2-independent effects of K27M mutation are crucial for tumor development. Importance of the Study: This study is the first to demonstrate that H3F3A K27M mutations drive a repressive transcriptome by modulating chromatin accessibility independently of H3K27 trimethylation in Diffuse Midline Glioma (DMG). By isolating the effects of H3.3 K27me3 loss from those of the K27M mutation, we identified common and unique genes and pathways affected by each. We found that genes uniquely deregulated by K27M showed increased chromatin accessibility and upregulated gene expression, unlike other gene subsets affected by PRC2 knockout. Importantly, we determined the PRC2-independent function of K27M is also essential for tumorigenesis, as xenografts of H3.3 K27M/PRC2 WT cell lines formed tumors, while H3.3WT/PRC2 WT and K27M/PRC2 knockout cells did not. This research builds upon and advances prior studies, such as those identifying EZH2 as a therapeutic target in H3.3K27M DMGs, by revealing critical new pathways for gliomagenesis. The translational significance lies in identifying novel therapeutic targets against this aggressive pediatric cancer.

First referral hospitals in low- and middle-income countries: the need for a renewed focus.

First referral hospitals (FRHs) are the hospitals closest to the community, which offer expertise or technologies to complement more widely available 'basic' ambulatory care or inpatient care. Despite having been a subject of interest in global health policy in the latter half of the 20th century, in more recent decades, they appear to have been overshadowed. This paper reviews what is understood by FRH, drawing on both academic and policy literature, complemented by specific country case studies. We undertook three reviews: a grey literature review of global and regional policy reports and documents, a structured review of the academic literature on FRH and a review of FRH-related policies in eight countries. Our findings indicate that there is confusion regarding the definitions and roles of FRH; they have fallen off the policy agenda globally and they suffer from lack of advocates in part related to the absence of cohesive definition. Meanwhile, these facilities continue to fulfil important functions in health systems in low- and middle-income countries, and expectations for service delivery remain high. In light of these findings, this paper calls for renewed interest and investment in FRH from the global health academic and policy-making community.

Determination of causes of adult deaths using minimally invasive tissue sampling in Gandaki province of Nepal: a multicenter hospital-based study.

BACKGROUND: Minimally Invasive Tissue Sampling (MITS) has been successfully used to establish the cause of death in low- and middle-income countries, mostly in stillbirths and neonates. The objective of this study was to determine the causes of death among adults using MITS in the Gandaki province of Nepal and to find out the contribution of MITS to identify the causes of death. METHODS: A multicentric hospital-based pilot study was conducted to enroll 100 cases of adult deaths. The specimens of cerebrospinal fluid, blood, brain, lungs, and liver tissue were collected utilizing MITS. These specimens underwent standard histopathological, serological, and microbiological analyses. The findings from MITS, and if available, clinical records and forensic autopsy findings were compiled and the cause of death panel identified the causes of death. The final cause of death allocated to each case was based on the WHO International Medical Certificate of Death. RESULTS: Among a total of 100 cases enrolled during the study period, infectious cause attributed to the immediate cause of death in 77 (77%), cardiovascular in 10 (10%), neurological in 8 (8%), malignancy in two (2%), and gastrointestinal and hepatobiliary cause in one (1%) case. The mean age of the cases was 50.8 ± 15.9 years and 76 (76%) were males. MITS established the cause of death in the causal chain of events in 81(81%) cases and identified the cause of death significantly more with infectious than non-infectious causes (p < 0.001). CONCLUSIONS: MITS was useful in establishing the cause of death in the majority of adult deaths and the most common cause was infectious disease. Our findings suggest that MITS can be a valuable and alternative tool for mortality surveillance in low-resource settings, where complete diagnostic autopsies are less accepted or less prioritized.

Knowledge, Attitudes, and Practices Concerning COVID-19 in Nepal.

BACKGROUND: Coronavirus disease-2019 (COVID-19) severely affects patients with chronic diseases. Adequate knowledge, attitudes, and practice related to COVID-19 is associated with decrease morbidity and mortality. Therefore, this aimed to assess knowledge, attitudes, and practice toward COVID-19 disease among chronic diseases patients visiting tertiary hospitals in Kathmandu. METHODS: A cross-sectional study was conducted among chronic disease patients who visited Tribhuvan University Teaching Hospital and Manmohan Cardiothoracic Vascular and Transplant Center, Kathmandu. Structured questionnaire was used to collect patients' socio-demographic data and perspectives on COVID-19. RESULTS: Four hundred chronic disease patients participated in the study, with 53.2% female and 46.8% male, and 56.8% of age ≥50 years. Overall, 55.5% of the participants had good knowledge, 56.2% had good practice, and 30.7% had positive attitude towards COVID-19. Younger patients, 18-34 years, were found to have higher odds of having good knowledge compared to other age groups (aOR: 2.5; 95% CI: 1.3-6.0). The patients with less than average family income and those unable to read and write had lower odds of having positive attitude towards COVID-19 (aOR: 0.6; 95% CI: 0.4-0.9 and aOR: 0.4; 95%CI: 0.2-0.9, respectively). A statistically significant correlation was found between the patients' knowledge and practice, knowledge and attitude, and attitude and practice (p <0.001). CONCLUSIONS: Nearly half of the chronic disease patients in Nepal had poor knowledge and practice whereas more than two-third had negative attitude towards COVID-19 disease. Older age of the patient was significantly associated with poor knowledge and practice.

Social determinants of health and the double burden of disease in Nepal: a secondary analysis.

BACKGROUND: As the global burden of disease evolves, lower-resource countries like Nepal face a double burden of non-communicable and infectious disease. Rapid adaptation is required for Nepal's health system to provide life-long, person-centred care while simultaneously improving quality of infectious disease services. Social determinants of health be key in addressing health disparities and could direct policy decisions to promote health and manage the disease burden. Thus, we explore the association of social determinants with the double burden of disease in Nepal. METHODS: This is a retrospective, ecological, cross-sectional analysis of infectious and non-communicable disease outcome data (2017 to 2019) and data on social determinants of health (2011 to 2013) for 753 municipalities in Nepal. Multinomial logistic regression was conducted to evaluate the associations between social determinants and disease burden. RESULTS: The 'high-burden' combined double burden (non-communicable and infectious disease) outcome was associated with more accessible municipalities, (adjOR3.94[95%CI2.94-5.28]), municipalities with higher proportions of vaccine coverage (adjOR12.49[95%CI3.05-51.09]) and malnutrition (adjOR9.19E103[95%CI19.68E42-8.72E164]), lower average number of people per household (adjOR0.32[95%CI0.22-0.47]) and lower indigenous population (adjOR0.20[95%CI0.06-0.65]) compared to the 'low-burden' category on multivariable analysis. 'High-burden' of non-communicable disease was associated with more accessible municipalities (adjOR1.93[95%CI1.45-2.57]), higher female proportion within the municipality (adjOR1.69E8[95%CI3227.74-8.82E12]), nutritional deficiency (adjOR1.39E17[95%CI11799.83-1.64E30]) and malnutrition (adjOR2.17E131[95%CI4.41E79-1.07E183]) and lower proportions of population under five years (adjOR1.05E-10[95%CI9.95E-18-0.001]), indigenous population (adjOR0.32[95%CI0.11-0.91]), average people per household (adjOR0.44[95%CI0.26-0.73]) and households with no piped water (adjOR0.21[95%CI0.09-0.49]), compared to the 'low-burden' category on adjusted analysis. 'High burden' of infectious disease was also associated with more accessible municipalities (adjOR4.29[95%CI3.05-6.05]), higher proportions of population under five years (adjOR3.78E9[95%CI9418.25-1.51E15]), vaccine coverage (adjOR25.42[95%CI7.85-82.29]) and malnutrition (adjOR4.29E41[95%CI12408.29-1.48E79]) and lower proportions of households using firewood as fuel (adjOR0.39[95%CI0.20-0.79]) ('moderate-burden' category only) compared to 'low-burden'. CONCLUSIONS: While this study produced imprecise estimates and cannot be interpreted for individual risk, more accessible municipalities were consistently associated with higher disease burden than remote areas. Female sex, lower average number per household, non-indigenous population and poor nutrition were also associated with higher burden of disease and offer targets to direct interventions to reduce the burden of infectious and non-communicable disease and manage the double burden of disease in Nepal.

Participatory Approach to Develop Evidence-Based Clinical Ethics Guidelines for the Care of COVID-19 Patients: A Mixed Method Study From Nepal.

During health emergencies such as the COVID-19 pandemic, healthcare workers face numerous ethical challenges while catering to the needs of patients in healthcare settings. Although the data recapitulating high-income countries ethics frameworks are available, the challenges faced by clinicians in resource-limited settings of low- and middle-income countries are not discussed widely due to a lack of baseline data or evidence. The Nepali healthcare system, which is chronically understaffed and underequipped, was severely affected by the COVID-19 pandemic in its capacity to manage health services and resources for needy patients, leading to ethical dilemmas and challenges during clinical practice. This study aimed to develop a standard guideline that would address syndemic ethical dilemmas during clinical care of COVID-19 patients who are unable to afford standard-of-care. A mixed method study was conducted between February and June of 2021 in 12 government designated COVID-19 treatment hospitals in central Nepal. The draft guideline was discussed among the key stakeholders in the pandemic response in Nepal. The major ethical dilemmas confronted by the study participants (50 healthcare professionals providing patient care at COVID-19 treatment hospitals) could be grouped into five major pillars of ethical clinical practice: rational allocation of medical resources, updated treatment protocols that guide clinical decisions, standard-of-care regardless of patient's economic status, effective communication among stakeholders for prompt patient care, and external factors such as political and bureaucratic interference affecting ethical practice. This living clinical ethics guideline, which has been developed based on the local evidence and case stories of frontline responders, is expected to inform the policymakers as well as the decision-makers positioned at the concerned government units. These ethics guidelines could be endorsed with revisions by the concerned regulatory authorities for the use during consequent waves of COVID-19 and other epidemics that may occur in the future. Other countries affected by the pandemic could conduct similar studies to explore ethical practices in the local clinical and public health context.

Anti-SARS-CoV-2 Antibody Level among Renal Transplant Recipients: A Case Report from Nepal.

Globally, SARS-CoV-2 has caused significant public health burden, mainly in patients with underlying comorbidities including both communicable and noncommunicable diseases. Solid organ transplant recipients under immunesupressive medication are also amongst the high risk group. There is only sparse data on immunity against SARS-CoV-2 infection among renal transplant recipients. In this case report, we present the level of anti-SARS-CoV-2 antibody of three kidney transplant recipients after vaccination against COVID-19 virus. All three cases had received two doses of Oxford-AstraZeneca COVID-19 vaccine AZD1222 (ChAdOx1). Serological analysis showed protective level of circulating antibodies in the blood of all three cases. Although two out of three patients in the study acquired COVID-19 infection after immunization, they recovered with mild clinical course. Hence, we conclude that despite immune-suppressed status of transplant recipients, COVID-19 vaccination could protect them against severe illness.

Childhood Invasive Bacterial Disease in Kathmandu, Nepal (2005-2013).

BACKGROUND: Invasive bacterial disease (IBD; including pneumonia, meningitis, sepsis) is a major cause of morbidity and mortality in children in low-income countries. METHODS: We analyzed data from a surveillance study of suspected community-acquired IBD in children <15 years of age in Kathmandu, Nepal, from 2005 to 2013 before introduction of pneumococcal conjugate vaccines (PCV). We detailed the serotype-specific distribution of invasive pneumococcal disease (IPD) and incorporated antigen and PCR testing of cerebrospinal fluid (CSF) from children with meningitis. RESULTS: Enhanced surveillance of IBD was undertaken during 2005-2006 and 2010-2013. During enhanced surveillance, a total of 7956 children were recruited of whom 7754 had blood or CSF culture results available for analysis, and 342 (4%) had a pathogen isolated. From 2007 to 2009, all 376 positive culture results were available, with 259 pathogens isolated (and 117 contaminants). Salmonella enterica serovar Typhi was the most prevalent pathogen isolated (167 cases, 28% of pathogens), followed by Streptococcus pneumoniae (98 cases, 16% pathogens). Approximately, 73% and 78% of pneumococcal serotypes were contained in 10-valent and 13-valent PCV, respectively. Most cases of invasive pneumococcal disease (IPD) were among children ≥5 years of age from 2008 onward. Antigen and PCR testing of CSF for pneumococci, Haemophilus influenzae type b and meningococci increased the number of these pathogens identified from 33 (culture) to 68 (culture/antigen/PCR testing). CONCLUSIONS: S. enterica serovar Typhi and S. pneumoniae accounted for 44% of pathogens isolated. Most pneumococcal isolates were of serotypes contained in PCVs. Antigen and PCR testing of CSF improves sensitivity for IBD pathogens.

Mitigating losses: how scientific organisations can help address the impact of the COVID-19 pandemic on early-career researchers.

Scientific collaborations among nations to address common problems and to build international partnerships as part of science diplomacy is a well-established notion. The international flow of people and ideas has played an important role in the advancement of the 'Sciences' and the current pandemic scenario has drawn attention towards the genuine need for a stronger role of science diplomacy, science advice and science communication. In dealing with the COVID-19 pandemic, visible interactions across science, policy, science communication to the public and diplomacy worldwide have promptly emerged. These interactions have benefited primarily the disciplines of knowledge that are directly informing the pandemic response, while other scientific fields have been relegated. The effects of the COVID-19 pandemic on scientists of all disciplines and from all world regions are discussed here, with a focus on early-career researchers (ECRs), as a vulnerable population in the research system. Young academies and ECR-driven organisations could suggest ECR-powered solutions and actions that could have the potential to mitigate these effects on ECRs working on disciplines not related to the pandemic response. In relation with governments and other scientific organisations, they can have an impact on strengthening and creating fairer scientific systems for ECRs at the national, regional, and global level.

Building Capacity and Infrastructure at Hospitals Implementing Minimally Invasive Tissue Sampling: Experience and Lessons Learned From Nepal, Rwanda, and Tanzania.

BACKGROUND: Minimally invasive tissue sampling (MITS) is a useful tool to determine cause of death in low- and middle-income countries (LMICs). In 2019 the MITS Surveillance Alliance supported the implementation of small-scale postmortem studies using MITS in several LMICs. METHODS: In this article we describe the preparations, challenges, and lessons learned as part of implementing MITS across 4 study sites in 3 countries: Nepal, Rwanda, and Tanzania. We describe the process for building capacity to conduct MITS, which consisted of training in MITS sample collection, individual site assessment to determine readiness and gaps prior to implementation, site visits as sites began implementation of MITS, and feedback based on remote evaluation of histology slides via an online portal. RESULTS: The 4 study sites each conducted 100 MITS, for a total of 400. All 4 sites lacked sufficient infrastructure and facilities to conduct MITS, and upgrades were required. Common challenges faced by sites included that clinical autopsies were neither routinely conducted nor widely accepted. Limited clinical records made cause of death determination more difficult. Lessons learned included the importance of sensitization of the community and medical staff to MITS to enhance understanding and increase consent. CONCLUSIONS: The study sites accomplished MITS and utilized the available support systems to overcome the challenges. The quality of the procedures was satisfactory and was facilitated through the organized capacity-building programs.

Cost Evaluation of Minimally Invasive Tissue Sampling (MITS) Implementation in Low- and Middle-Income Countries.

BACKGROUND: Low- and middle-income countries (LMICs) face disproportionately high mortality rates, yet the causes of death in LMICs are not robustly understood, limiting the effectiveness of interventions to reduce mortality. Minimally invasive tissue sampling (MITS) is a standardized postmortem examination method that holds promise for use in LMICs, where other approaches for determining cause of death are too costly or unacceptable. This study documents the costs associated with implementing the MITS procedure in LMICs from the healthcare provider perspective and aims to inform resource allocation decisions by public health decisionmakers. METHODS: We surveyed 4 sites in LMICs across Sub-Saharan Africa and South Asia with experience conducting MITS. Using a bottom-up costing approach, we collected direct costs of resources (labor and materials) to conduct MITS and the pre-implementation costs required to initiate MITS. RESULTS: Initial investments range widely yet represent a substantial cost to implement MITS and are determined by the existing infrastructure and needs of a site. The costs to conduct a single case range between $609 and $1028 per case and are driven by labor, sample testing, and MITS supplies costs. CONCLUSIONS: Variation in each site's use of staff roles and testing protocols suggests sites conducting MITS may adapt use of resources based on available expertise, equipment, and surveillance objectives. This study is a first step toward necessary examinations of cost-effectiveness, which may provide insight into cost optimization and economic justification for the expansion of MITS.

Burden of Serious Bacterial Infections and Multidrug-Resistant Organisms in an Adult Population of Nepal: A Comparative Analysis of Minimally Invasive Tissue Sampling Informed Mortality Surveillance of Community and Hospital Deaths.

BACKGROUND: Bacterial diseases are the leading cause of mortality globally, and due to haphazard use of antibiotics, antimicrobial resistance has become an emerging threat. METHODS: This cross-sectional observational study utilized a minimally invasive tissue sampling procedure to determine the cause of death among an adult population. Bacterial cultures (blood, cerebrospinal fluid, lung tissue) and antibiotic susceptibility were evaluated, and the results were compared between community and hospital deaths. RESULTS: Of 100 deceased persons studied, 76 (76%) deaths occurred in the community and 24 (24%) in the hospital. At least 1 bacterial agent was cultured from 86 (86%) cases; of these, 74 (86%) had a bacterial disease attributed as the primary cause of death, with pneumonia (35, 47.3%), sepsis (33, 44.6%), and meningitis (3, 4.1%) most common. Of 154 bacterial isolates (76.6% from the community and 23.4% from the hospital) detected from 86 culture-positive cases, 26 (16.8%) were multidrug-resistant (MDR). Klebsiella species were the most common (13 of 26) MDR organisms. The odds of getting an MDR Klebsiella infection was 6-fold higher among hospital deaths compared with community deaths (95% confidence interval [CI], 1.37-26.40; P = .017) and almost 23-fold higher (CI, 2.45-213.54; P = .006) among cases with prior antibiotic use compared to those without. CONCLUSIONS: High incidence of serious bacterial infections causing death of adults in the community, with most MDR organisms isolated from hospitalized cases, calls for robust surveillance mechanisms and infection prevention activities at the community level and evidence-driven antibiotic stewardship in healthcare settings.

Correction: Mitigating losses: how scientific organisations can help address the impact of the COVID-19 pandemic on early-career researchers.

[This corrects the article DOI: 10.1057/s41599-021-00944-1.].

Rheumatic Heart Disease among Pregnant Women with Cardiac Diseases in a Tertiary Care Center of Nepal: A Descriptive Cross-sectional Study.

INTRODUCTION: Cardiac disease in pregnancy is a major cause of maternal mortality and morbidity in women, particularly in resource limited countries like Nepal. Rheumatic Heart Disease is the commonest cardiac disease complicating pregnancy. There is very limited data and evidence from Nepal regarding rheumatic heart disease complicating the pregnancy. The study aims to find out the prevalence of rheumatic heart disease among cardiac disease patients in a tertiary care hospital. METHODS: A descriptive cross-sectional study was conducted among 41 women with cardiac disease who delivered babies at Chitwan Medical College from 1st January 2018 to 31st December 2019, after taking ethical approval from the Institutional Review Committee. A convenient sampling method was used. Statistical Package for the Social Sciences was used for data analysis. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: Among 41 pregnant women with cardiac disease, 32 (78%) (95% Confidence Interval = 65.32-90.68) had rheumatic heart disease. The mean age of the affected pregnant women was 24.9±4.49 years. Out of 32 patients with rheumatic heart disease, postpartum haemorrhage was the most common maternal complication 5 (15.6%) followed by hypertension 4 (9.7%). CONCLUSIONS: Rheumatic Heart Disease was highly common among pregnant women with cardiac disease.

Maternal and fetal characteristics and causes of stillbirth in a tertiary care hospital of Nepal: secondary analysis of registry-based surveillance data.

OBJECTIVES: Stillbirth is one of the vital indicators of quality care. This study aimed to determine maternal-fetal characteristics and causes of stillbirth in Nepal. DESIGN: Secondary analysis of single-centred registry-based surveillance data. SETTING: The study was conducted at the Department of Obstetrics and Gynecology, Chitwan Medical College Teaching Hospital, a tertiary care hospital located in Bharatpur, Nepal. PARTICIPANTS: All deliveries of intrauterine fetal death, at or beyond 22 weeks' period of gestation and/or birth weight of 500 g or more, conducted between 16 July 2017 and 15 July 2019 were included in the study. MAIN OUTCOME MEASURES: The primary outcome measure of this study was stillbirth, and the secondary outcome measures were maternal and fetal characteristics and cause of stillbirth. RESULTS: Out of 5282 institutional deliveries conducted over 2 years, 79 (1.5%) were stillbirths, which gives the stillbirth rate of 15 per 1000 births. Of them, the majority (75; 94.9%) were vaginal delivery and only four (5.1%) were caesarean section (p<0.0001). The proportion of the macerated type of stillbirth was more than that of the fresh type (58.2% vs 41.8%; p=0.13). Only half of the mothers who experienced stillbirth had received antenatal care. While the cause of fetal death was unknown in one-third of cases (31.6%; 25/79), among likely causes, the most common was maternal hypertension (29.1%), followed by intrauterine infection (8.9%) and fetal malpresentation (7.6%). Four out of 79 stillbirths (5%) had a birth defect. CONCLUSION: High rate of stillbirths in Nepal could be due to the lack of quality antenatal care. The country's health systems should be strengthened so that pregnancy-related risks such as maternal hypertension and infections are identified early on. Upgrading mothers' hygiene and health awareness is equally crucial in reducing fetal deaths in low-resource settings.

Characterizing Genetic Regulatory Elements in Ovine Tissues.

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

Combination of convalescent plasma therapy and repurposed drugs to treat severe COVID-19 patient with multimorbidity.

Combination of convalescent plasma therapy and repurposed drugs such as dexamethasone and remdesivir could be beneficial for severe COVID-19 patients with obesity and chronic diseases such as diabetes and hypertension.

Disseminated cryptococcosis in a deceased with HIV-1 diagnosed by minimally invasive tissue sampling technique.

Minimal invasive tissue sampling (MITS) technique detected HIV infection and disseminated cryptococcosis in an adult female with sudden death. A proper autopsy is essential to diagnose the exact cause of death and MITS can suffice in natural deaths.