Delving the vitamin D receptor variation and expression profiles in the context of type 2 diabetes among families.

BACKGROUND: Vitamin D is essential for insulin secretion and sensitivity. Consequently, its inadequacy is linked to higher insulin resistance and Type 2 Diabetes (T2D). The Vitamin D receptor (VDR) gene is one potential candidate for T2D, and multiple polymorphisms in VDR have been examined in various populations, but no conclusive answers have been provided. OBJECTIVES: This study was designed to evaluate the susceptibility of VDR gene polymorphism and its expression in diabetic families in Pakistan. METHODOLOGY: In this family-based study, twenty diabetic families with a positive family history of T2D and at least three T2D patients were recruited from outpatient clinics and public hospitals. The current study comprised 143 individuals with 55 affected and 88 unaffected individuals. Blood samples of the selected families were collected. DNA was extracted from the collected samples and the PCR-RFLP method was followed to identify the genotyping and RT-qPCR for expression. Phenotypic and genotypic pedigrees of the families were developed by the progeny online tool. The association values of SNPs were determined by TDT and DFAM analysis implemented on Plink software. RESULTS: The results explained a significant familial aggregation among phenotypic characters including Age, Gender, BMI (body mass index), age of disease diagnosis, disease duration, and blood pressure in the probands, affected FDRs (First Degree Relatives) and affected SDRs (Second Degree Relatives). A significant association of rs731236 C/T (OR = 1.522), rs2228570 C/T (OR = 1.327) with p < 0.05. Whereas, for rs1544410 G/A (OR = 0.9706) and rs7975232 T/G (OR = 0.7368) no considerable association evidence was seen (p > 0.05) in families. The mRNA expression of VDR increased threefold (p = 0.0204) in patients compared to controls. Variation-based expression analysis exhibited that the rs2228570 genotype influences the expression. CONCLUSION: A linkage was found among the FDRs with probands. Variation in the gene VDR at loci rs731236 and rs2228570 was associated with familial T2D. However further research is required to explore more genetic factors that could influence T2D risks in families.

Prediction of putative epitope-based vaccine against all corona virus strains for the Chinese population: Approach toward development of vaccine.

Currently, the whole world is facing the coronavirus disease-19 pandemic. As of now, approximately 0.15 million people around the globe are infected with the novel coronavirus. In the last decade, two strains of the coronavirus family, severe acute respiratory syndrome-related coronavirus and Middle East respiratory syndrome coronavirus, also resulted in epidemics in south Asian and the Middle Eastern countries with high mortality rate. This scenario demands the development of a putative vaccine which may provide immunity against all current and new evolving coronavirus strains. In this study, we designed an epitope-based vaccine using an immunoinformatic approach. This vaccine may protect against all coronavirus strains. The vaccine is developed by considering the geographical distribution of coronavirus strains and host genetics (Chinese population). Nine experimentally validated epitopes sequences from coronavirus strains were used to derive the variants considering the conservancy in all strains. Further, the binding affinities of all derived variants were checked with most abundant human leukocyte antigen alleles in the Chinese population. Three major histocompatibility complex (MHC) Class I epitopes from spike glycoprotein and nucleoprotein showed sufficient binding while one MHC Class II epitope from spike glycoprotein was found to be an effective binder. A cocktail of these epitopes gave more than 95% population coverage in the Chinese population. Moreover, molecular dynamics simulation supported the aforementioned predictions. Further, in vivo studies are needed to confirm the immunogenic potential of these vaccines.

Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection.

Hepatitis C virus (HCV) entry into isolated primary liver cells and cell lines requires interaction with the cell surface receptors. The study of HCV attachment with host cell surface receptors has been hindered by the unavailability of competent cell culture based system for HCV propagation. This problem has been overcome by the development of genetically tagged infectious HCV pseudo particles (HCVpp) harboring unmodified E1 and E2 glycoproteins. Studies using cell binding assays together with infection assays using HCVpp have shown that CD81 and scavenger receptor (SRBI) are actively involved in binding with envelope proteins facilitating the viral entrance process. This paper aimed to develop HCVpp of local HCV 3a Pakistani isolate and to study the viral tropism role of CD81 and SRBI receptors in HCV infectivity. HCV E1 and E2 genes were amplified and cloned in mammalian expression vector pcDNA 3.1/myc. The expressing plasmid of HCV E1-E2 glycoprotein in native form was co-transfected into 293FT cells with lentiviral packaging plasmid encoding the MLV Gag-Pol core proteins, and a packaging competent MLV-derived genome (pMLVYCMV-Luc) encoding the luciferase marker protein to produce infectious HCVpp. Anti-CD81 antibody (CBL579), anti-SRBI type II antibody (sc-20441) HCV anti-E2 mouse IgG1 (sc-65457) and HCV anti-E1 antibody mouse IgG1 (sc-65459) were used in this setup. We showed that primary site of viral replication is liver which involve CD81 and SRBI receptors for HCV gp-dependent infection with HCVpp. This is the preliminary reported cell cultured based mechanism from Pakistan which facilitated functional studies of different antiviral agents. Understanding of this technique will help in development of new antiviral therapeutics focusing on earlier steps of HCV life cycle. We have developed infectious pseudo particles of local 3a-isolate and concluded that a number of liver-specific surface proteins function along with CD81 and SRBI receptor regarding HCV infectivity. To endeavors and to identify this liver specific co-receptor molecule(s) will provide insights into the role of these molecules in the initial steps of HCV life cycle.

Association of epilepsy and neurological impairments with homozygous recessive missense mutations found in the genes responsible for ganglioside biosynthesis (ST3GAL5) and calcium voltage-gated channels (CACNA1H) - insights through molecular dynamic simulations.

With over 2.2 million cases, the incidence rate of epilepsy in Pakistan is far higher than the rest of the world due primarily to the frequent, traditionally imposed cousin marriages. In the present study, comprehensive whole exome sequencing (WES) analyses of a three-generation family with four affected members presenting 'unexplained' childhood absence epilepsy (CAE), seizures and dementia, was performed in a quest to identify heritable, epilepsy-causal gene variants to better aid in carrier screening and genetic counselling. The WES data was generated, analyzed, and validated through Sanger's sequencing, molecular dynamic simulation (MDS) analysis, and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) studies. Two homozygous recessive, missense mutations in ST3GAL5 (c.311A > G, p. His104Arg) and CACNA1H (c.6230G > A, p. Arg2077His) genes, earlier regarded as benign or of uncertain significance, have been identified as a potential etiology. Comparative MDS and free binding energy calculations revealed substantial structural perturbations in mutant forms of ST3GAL5 leading to decreased binding and reduced catalytic activity of the p.His104Arg and two other functional variants (p.Val74Glu and p.Arg288Ter) when compared with wild type. Our findings reinforce that WES analyses may uncover 'hidden', heritable variants and together with MDS and MM/GBSA may provide plausible clues to answer the unexplained causes of epilepsy for an effective management and better patient outcome. Further, revisit of epilepsy-associated mutational landscape in population context is imperative as the variants with 'benign' tags may turn out to be 'non-benign', when exist in combination with other benign.Communicated by Ramaswamy H. Sarma.

Hepatitis C virus transmission cluster among injection drug users in Pakistan.

Hepatitis C virus (HCV) infections are public health problem across the globe, particularly in developing countries. Pakistan has the second highest prevalence of HCV infection worldwide. Limited data exist from Pakistan about persons who inject drugs (PWID) and are at significant risk of exposure to HCV infection and transmission. Serum specimens (n = 110) collected from PWID residing in four provinces were tested for molecular markers of HCV infection. Next generation sequencing (NGS) of the hypervariable region (HVR1) of HCV and Global Hepatitis Outbreak and Surveillance Technology (GHOST) were used to determine HCV genotype, genetic heterogeneity, and construct transmission networks. Among tested specimens, 47.3% were found anti-HCV positive and 34.6% were HCV RNA-positive and belonged to four genotypes, with 3a most prevalent followed by 1a, 1b and 4a. Variants sampled from five cases formed phylogenetic cluster and a transmission network. One case harbored infection with two different genotypes. High prevalence of infections and presence of various genotypes indicate frequent introduction and transmission of HCV among PWID in Pakistan. Identification of a transmission cluster across three provinces, involving 20% of all cases, suggests the existence of a countrywide transmission network among PWIDs. Understanding the structure of this network should assist in devising effective public health strategies to eliminate HCV infection in Pakistan.