Whole Genome Sequence Analysis to Identify SARS-CoV-2 Variant in Nepal.

Background The spread of SARS-CoV-2 has become a global public health crisis. Nepal is facing the second wave of COVID-19 pandemic but, there is still a limited data on the genomic sequence of SARS-CoV-2 variants circulating in Nepal. Objective The objective of this study is to sequence the whole genome of SARS-CoV-2 in Nepal to detect possible mutation profiles and phylogenetic lineages of circulating SARSCoV-2 variants. Method In this study, swab samples tested positive for SARS-CoV-2 were investigated. After RNA extraction, the investigation was performed through real-time PCR followed by whole genome sequencing. The consensus genome sequences were, then, analyzed with appropriate bioinformatics tools. Result Sequence analysis of two SARS-CoV-2 genomes from patient without travel history (Patient A1 and A2) were found to be of lineage B.1.1. Similarly, among other four samples from subjects returning from the United Kingdom, genomes of two samples were of lineage B.1.36, and the other two were of lineage B.1.1.7 (Alpha Variant). The mutations in the consensus genomes contained the defining mutations of the respective lineages of SARS-CoV-2. Conclusion We confirmed two genomic sequences of variant of concern VOC-202012/01 in Nepal. Our study provides the concise genomic evidence for spread of different lineages of SARS-CoV-2 - B.1.1, B.1.36 and B.1.1.7 of SARS-CoV-2 in Nepal.

Evaluation of usefulness of a commercial agarose gel electrophoresis kit (QuickGel SP) for bovine serum protein electrophoresis.

The aim of this study was to show the usefulness of a commercial agarose gel electrophoresis (AGE) kit (QuickGel SP) for separating bovine serum protein fractions in comparison with conventional cellulose acetate electrophoresis (CAE). Serum protein bands were verified using five reference reagents corresponding to albumin and α1-, ß1-, ß2-, and γ-globulins. AGE clearly revealed six separated fractions of albumin and α1-, α2-, ß1-, ß2-, and γ-globulin fractions in 100% and 77.8% in serum samples of dairy cows from the healthy (n=27) and diseased groups (n=27), respectively. The α1- and α2-globulins were not separated by CAE in 14.8% and 96.3% of the samples from the healthy and diseased groups, respectively, whereas ß2- and γ-globulin were not separated by CAE in 96.3% and 100% of the samples from the healthy and diseased groups, respectively. More than 94% of the points for the α-globulin fractions (α1- and α2-globulins), the ß-γ-globulin fractions (ß1-, ß2-, and γ-globulins), and the albumin/globulin ratio between AGE and CAE were within agreement on the Bland-Altman plots. However, the mean biases were not near zero in the albumin and ß-γ-globulin fractions. These results suggest that the high-resolution commercial AGE kit can be utilized to separate bovine serum protein fractions.

Molecular and phylogenetic analyses of the liver amphistome Explanatum explanatum (Creplin, 1847) Fukui, 1929 in ruminants from Bangladesh and Nepal based on nuclear ribosomal ITS2 and mitochondrial nad1 sequences.

Explanatum explanatum flukes, liver amphistomes of ruminants, cause significant economic loss in the livestock industry by inducing severe liver damage. A total of 66 flukes from 26 buffaloes and 7 cattle in four different geographic areas of Bangladesh and 20 flukes from 10 buffaloes in the Chitwan district of Nepal were subjected for analysis. The sequences (442 bp) of the second internal transcribed spacer (ITS2) of ribosomal DNA and the variable fragments (657 bp) of mitochondrial nicotinamide dehydrogenase subunit 1 (nad1) of E. explanatum flukes from Bangladesh and Nepal were analysed. The aim of this study was molecular characterization of the flukes and to elucidate their origin and biogeography. In the ITS2 region, two genotypes were detected among the flukes from Bangladesh, while flukes from Nepal were of only one genotype. Phylogenetic analyses inferred from the nad1 gene revealed that at least four divergent populations (groups I-IV) are distributed in Bangladesh, whereas two divergent populations were found to be distributed in Nepal. Fst values (pairwise fixation index) suggest that Bangladeshi and Nepalese populations of group I to IV are significantly different from each other; but within groups III and IV, the populations from Bangladesh and Nepal were genetically close. This divergence in the nad1 gene indicates that each lineage of E. explanatum from diverse geography was co-adapted during the multiple domestication events of ruminants. This study, for the first time, provides molecular characterization of E. explanatum in Bangladesh and Nepal, and may provide useful information for elucidating its origin and dispersal route in Asia.

Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever.

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor ß (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

Involving diaspora and expatriates as human resources in the health sector in Nepal.

BACKGROUND: Health professional mobility has increased in the recent years and is one of the public health concerns in the developing countries including Nepal. On the other hand, we can't ignore a positive shift of Nepali diaspora coming back to Nepal for some work related projects. The objective of this study was thus to estimate the number of Nepalese Diaspora and foreign expatriate those are coming to Nepal and explore the ways and process of their engagement in the health sector of Nepal. METHODS: Mixed method was used. In total, 13 Key Informant Interviews were conducted at the central level along with record review from professional councils. RESULTS: Nepalese Diasporas mainly come through Diaspora Volunteering Organizations, Non Resident Nepali Association and personal connections to the place of their origin. Nepalese Diasporas have supported as health specialists, health camps and project organizers, trainer and hospital promoters, supplier of equipment including ambulances etc. The Nepalese Diasporas are unrecorded with professional organizations such as NMC and NHPC. As such the real status and results of support from Nepalese Diaspora are not known. Overall, 5,120 foreign medical professionals have served to Nepal through NMC followed by 739 nursing professionals through NNC and 189 paramedical staff through NHPC as of 2012. CONCLUSIONS: Systematic information on number and characteristics of the Nepalese Diaspora and their role in the health sector of Nepal is limited. The health professional bodies have some record systems but they lack uniformity and systematic process.

Health governance at local level from human resource for health perspectives: the case of Nepal.

BACKGROUND: Evidence about effects of good governance in Human Resources for Health (HRH) is scant in Nepal. The study aimed to explore the situation of health governance at the local level and suggest measures to address the HRH challenges. METHODS: Ninety health facilities from Siraha, Bardiya and Doti districts were included in the study. Focus group discussions (N=36) with different groups and key informants interviews (33 VDC Secretaries, 76 Health Facility Management Committees and 9 central level policy makers and managers) were conducted. RESULTS: Only 49 (54%) of the health facilities have properly displayed signboard, 42 (47%) citizen charter, 36 (40%) free health services and Information on Aama program in 25 (28%) health facilities. In total 52 (58%) health facilities have not displayed names of women receiving Aama benefits. Seventy two out of 90 health facilities have not displayed social audit reports and 80 (89%) of the health facilities have not maintained complaint box. The initiative of decentralized human resource management, where implemented, has increased ownership at the local level. Staff retention has been reported well though it does not apply in case of the medical doctors. Rule of law in terms of human resource recruitment and transfer, promotion, and training were not fully implemented and were lenient in the upper level. Nepotism and power exercise was frequently reported as a hindrance in implementing the gender and social inclusion policy fully. CONCLUSIONS: Transparency, gender and social inclusion is yet to be implemented fully at the district and health facility level.

Effects of cortisol secreted via a 12-h infusion of adrenocorticotropic hormone on mineral homeostasis and bone metabolism in ovariectomized cows.

To evaluate the effects of endogenously secreted cortisol on mineral homeostasis and bone metabolism in cows, 4 ovariectomized Holstein cows were infused for 12 h with either an adrenocorticotropic hormone (ACTH) solution (0.5 mg/2 L isotonic NaCl solution per cow) or isotonic NaCl solution in a 2×2 crossover design. ACTH infusion stimulated cortisol secretion and increased plasma cortisol concentrations for 18 h (P<0.001), leading to an elevated plasma glucose concentration until 36 h (P<0.001). Plasma calcium and magnesium concentrations in ACTH-infused cows fluctuated within normal ranges, whereas hypophosphatemia was observed unequivocally. The biochemical bone resorption markers tartrate-resistant acid phosphatase 5b and hydroxyproline decreased following ACTH infusion (P<0.001 and P=0.003, respectively). Similarly, the bone formation marker, bone-specific alkaline phosphatase, decreased continuously until 72 h after the ACTH infusion (P<0.001). These results demonstrate that increased secretion of cortisol via a 12-h ACTH infusion disrupted homeostasis of inorganic phosphate and suppressed bone metabolism in ovariectomized cows without involving gonadal steroid hormones.

Effects of heavy metals (Hg2+, cd2+, Pb2+) during the embryonic development of acridid grasshoppers (Insecta, caelifera)

Heavy metals present in the soil get differently accumulated in organisms and show different rates of toxicity at different stages of the life cycle of any organism living there. To see whether such toxicants get accumulated during the embryonic development and/or change the normal developmental processes of organisms exposed to heavy metals, freshly laid eggs and egg pods of two acridids, Aiolopus thalassinus and Eyprepocnemis plorans, were tested against different concentrations of Hg2+, Cd2+, and Pb2+. At increasing Hg2+ concentration in the substrate, an increased egg mortality and reduced nymphal hatching in A. thalassinus were observed, yet no change in the normal duration for embryonic development was noticed. A higher hatching rate of E. plorans nymphs than that of A. thalassinus could possibly be due to the higher tolerance, contributed by larger egg volume and thicker foam around the egg pods of the former. Treated concentrations of Hg2+ could be of sublethal (0.121 &mgr;g Hg2+/g substrate) to lethal (0.605 &mgr;g and more Hg2+/g substrate) doses. The mortality of eggs did not always increase with increasing substrate concentration of Cd2+ and Pb2+, and the hatching rates of both grasshopper species in many cases were even higher than that in the control. Still, lower accumulation factors of Cd2+ and Pb2+ than that of Hg2+ were found coupled with not increased mortality. The tested concentrations of Cd2+ and Pb2+ in the substrates, thus, could be of subacute doses. The embryonic development was found prolonged due to Cd2+ and Pb2+. During parallel egg and egg pod treatments, higher metal concentrations were found in not developed eggs than that in fresh nymphs hatching from the same substrates. This indicated the role of foam around the egg pods as "protective filter" against the toxic substances in the soil.