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Microbial populations play a crucial role in human health and the development of many diseases. These diseases often arise from the explosive proliferation of opportunistic bacteria, such as those in the nasal cavity. Recently, there have been increases in the prevalence of these opportunistic pathogens displaying antibiotic resistance. Thus, the study of the nasal microbiota and its bacterial diversity is critical in understanding pathogenesis and developing microbial-based therapies for well-known and emerging diseases. However, the isolation and analysis of these populations for clinical study complicates the already challenging task of identifying and profiling potentially harmful bacteria. Existing methods are limited by low sample throughput, expensive labeling, and low recovery of bacteria with ineffective removal of cells and debris. In this study, we propose a novel microfluidic channel with a zigzag configuration for enhanced isolation and detection of bacteria from human clinical nasal swabs. This microfluidic zigzag channel separates the bacteria from epithelial cells and debris by size differential focusing. As such, pure bacterial cell fractions devoid of large contaminating debris or epithelial cells are obtained. DNA sequencing performed on the separated bacteria defines the diversity and species present. This novel method of bacterial separation is simple, robust, rapid, and cost-effective and has the potential to be used for the rapid identification of bacterial cell populations from clinical samples.

BackgroundReceptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD.MethodsWe assessedRIPK1expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patientsviaimmunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, usingRipk1S25D/S25Dkinase-deficient mice and the RIPK1 kinase inhibitor GSK′547.ResultsRIPK1expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increasedRipk1expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition.ConclusionsRIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.

The COVID-19 pandemic heightened anti-Asian racism towards East Asian diasporas in North America. Experiences of racism encountered by East Asian communities have been documented to negatively impact their mental health.A scoping review was undertaken following Arksey and O'Malley's (2005) methodology to (a) map the foci of literature on racism and the mental health of East Asian diasporas in North America and (b) identify gaps in the current literature.A total of 1309 articles were identified in May 2021. Based on the inclusion criteria, 35 records were included. Two distinct mental health foci were found: mental health outcomes and mental healthcare access and utilization. The majority (The expanding knowledge base on COVID-19-related racial discrimination is reminiscent of previous literature examining the history of anti-Asian racism in North America. Greater attention is needed to navigate impactful anti-racism solutions for East Asian populations' mental health in North America.

Many important cellular processes involve protein–protein interactions (PPIs) mediated by a Short Linear Motif (SLiM) in one protein interacting with a globular domain in another. Despite their significance, these domain-motif interactions (DMIs) are typically low affinity, which makes them challenging to identify by classical experimental approaches, such as affinity pulldown mass spectrometry (AP-MS) and yeast two-hybrid (Y2H). DMIs are generally underrepresented in PPI networks as a result. A number of computational methods now exist to predict SLiMs and/or DMIs from experimental interaction data but it is yet to be established how effective different PPI detection methods are for capturing these low affinity SLiM-mediated interactions. Here, we introduce a new computational pipeline (SLiM-Enrich) to assess how well a given source of PPI data captures DMIs and thus, by inference, how useful that data should be for SLiM discovery. SLiM-Enrich interrogates a PPI network for pairs of interacting proteins in which the first protein is known or predicted to interact with the second protein via a DMI. Permutation tests compare the number of known/predicted DMIs to the expected distribution if the two sets of proteins are randomly associated. This provides an estimate of DMI enrichment within the data and the false positive rate for individual DMIs. As a case study, we detect significant DMI enrichment in a high-throughput Y2H human PPI study. SLiM-Enrich analysis supports Y2H data as a source of DMIs and highlights the high false positive rates associated with naïve DMI prediction. SLiM-Enrich is available as an R Shiny app. The code is open source and available via a GNU GPL v3 license at: https://github.com/slimsuite/SLiMEnrich. A web server is available at: http://shiny.slimsuite.unsw.edu.au/SLiMEnrich/.

Autosomal recessive congenital hereditary endothelial dystrophy (CHED2) is a recessively inherited eye disorder that is more common in consanguineous populations.Two families affected with CHED2 were recruited from the Punjab province of Pakistan to identify the underlying genetic defect. Blood samples from both the families, designated as CH01 and CH02, were collected. Genomic DNA was isolated. Initially, linkage analysis using microsatellite markers was carried out to confirm the linkage to the SLC4A11 gene, previously reported to be implicated in the pathology of the disease. Later on, sequencing was carried out to find the pathogenic mutation in the enrolled families. Identified variation was further confirmed by typing 50 ethnically matched normal control samples.The results of linkage analysis indicated the putative linkage to SLAC4A11 gene, located at the CHED2 locus on chromosome 20p13-p12 in both families. Mutational analysis revealed an unidentified homozygous mutation c.2024AC (p.E675A) in the affected members of both the families. Haplotype analysis of both the families showed that the affected members carry the same haplotype, thereby indicating a possibility of a common ancestral mutation. Use of bioinformatic tools including PolyPhen and SIFT suggested that a single amino acid change of E to A at position 675 affected the function of the protein.This study reports a newly identified mutation (c.2024AC) in the SLC4A11 gene segregating with the diseased haplotype in two consanguineous Pakistani families.

Objective To find potential peptide inhibitors against the NS2B/NS3 protease of DENV which in turn, can inhibit the viral replication inside host cell. Methods Cyclic peptides were designed having combination of positively charged amino acids using ChemSketch software and were converted to 3D structures. DENV NS3 protein structure was retrieved from Protein Data Bank (PDB) using PDB Id: 2FOM. DENV NS3 and cylic peptides were docked using MOE software after structural optimization. Results Through molecular docking it was revealed that most of the peptides bound deeply in the binding pocket of DENV NS2B/NS3 protease an had interactions with catalytic triad. Peptide 2 successfully blocked the catalytic triad of NS2B/NS3 protease. Peptide 1, 4 and 6 also had potential interactions with active residues of the NS2B/NS3 protease while all other peptides were in close contact with the active sites of NS2B/NS3 protease thus, these peptides can serve as a potential drug candidate to stop viral replication. Conclusions Thus, it can be concluded from the study that these peptides could serve as important inhibitors to inhibit the viral replication and need further in-vitro investigations to confirm their efficacy.

Hepatitis C virus (HCV) is a dreadful viral disease, responsible for 170 million cases worldwide, of which most are from Asia and Africa and approximately 10 million people are from Pakistan. Currently, the pegylated interferon alpha (PEG-INF-α) has been approved as the standard of care in combination with ribavirin and Boceprevir/Telaprevir. Many studies regarding gene expression analysis of liver biopsy samples of patients with chronic HCV infection have been carried out previously. However, there are very few reports of expression analysis carried out using blood samples of HCV patients. Therefore, in this study, gene expression of human immune responsive genes (MMP-9, OAS1) and fibrogenic responsive gene (KRT19) was done in the peripheral blood mononuclear cells (PBMCs) of chronic HCV infected patients having differences in viral titers. Blood samples were collected from different hospitals in Pakistan. RNA was extracted and cDNA was synthesized according to the protocol prescribed by the Enzynomics™ M-MLV Reverse Transcriptase(®) Kit. The synthesized cDNA was amplified through polymerase chain reaction (PCR) using specific primers of immune responsive genes. The results were further evaluated using real-time PCR. There was a significant increase in the expression of the immune responsive genes (MMP-9, OAS1, CXCL6, CXCR3, ApoA1, and MYC) of HCV genotype 3a patients compared to controls. Similarly, the expression of the fibrosis genes was upregulated in HCV genotype 3a patients compared to controls. The information gained through this study is helpful to identify a noninvasive marker to determine liver fibrosis, and may also give useful information to understand HCV pathogenesis and develop better therapeutic regimens.

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy.

The family of AP2/ERF plant specific transcription factors, including dehydration-responsive element binding (DREB) proteins, has been reported to activate and bind with the dehydration responsive element/C repeat (DRE/CRT) of stress-inducible gene promoters. We amplified DREB1A from Oryza sativa var. IR6 (807 bp) and subjected it to protein homology modelling and phylogenetic analysis. BLAST sequence analysis indicated high similarity (99%) with the O. sativa japonica group. Phylogenetic analysis was carried out with other 12 DREB sequences from cereals/grasses using Clustal Omega. Protein secondary structure and 3D models were determined using SOPMA and MODELLER v9.10, respectively. PROSA was used to determine the excellence and consistency of models. The results indicated that the Z-score value was -2.94 and 79.5% of the residues were found in the favoured region. A psi/phi Ramachandran plot was determined using PROCHECK. The molecular docking of the GCC-box binding domain (GBD) of the protein under investigation was done using atomic coordinates of Arabidopsis thaliana GBD- and GCC-box containing DNA, respectively. The tertiary structure of the modelled protein and template were found to be very similar as predicted by a root mean square deviation of 0.968 Å. It seemed that both the proteins interacted with the major groove of DNA using Beta-sheets. We attempted to expose the molecular basis of O. sativa var. IR6 DREB1A protein interaction with the target promoter sequence. The results highlighted that this gene could be a good candidate for production of abiotic stress-tolerant crop(s) in the future.

The dengue virus (DENV) genome encodes 10 different genes including the NS3 gene, which has a protease and helicase domain used in virus replication. This domain is a potential target for antiviral agents against dengue. Due to a high mutation rate, DENV is classified into four major serotypes (DENV1–DENV4). This study was designed to perform conservancy analysis of all four serotypes by drawing a consensus sequence for each serotype and then drawing a global consensus sequence to study conserved residues in all four serotypes. A total of 127 NS3 sequences belonging to all four serotypes were retrieved and aligned using multiple alignment feature of CLC Workbench and were subjected to phylogenetic tree construction. Conservancy analysis of NS3 revealed conserved peptides with active site residues that can be important in developing antiviral agents against dengue virus. Among conserved residues, residues G142, Ser144, and G145 (catalytic pocket residues), A219, D220, and D221 (divalent cations binding residues), and His56, Asp79, Ser144, 146 were highly conserved among all the serotypes. Residues from L138 to L149 and from L226 to L245 were also considerably conserved in all serotypes, while lysine141 mutated to serine in serotype 3. A total of 14 peptides from the conserved regions of DENV NS3 protein were identified, which may be helpful to develop peptide inhibitors. The DENV NS3 phylogenetic tree showed the evolutionary relationship among all four serotypes, and all serotypes of dengue were found to have evolved from the dengue 4 serotype. Because of its high variability, DENV has become a global health concern. It is important to study residues that are present in protease, helicase, the catalytic pocket Mg2+ binding site, and the AAA domain. This study revealed peptides with active site residues that are highly conserved among all four serotypes. These regions of the NS3 sequence may be helpful in developing antiviral agents.

Dengue virus infection has become a global threat affecting around 100 countries in the world. Currently, there is no licensed antiviral agent available against dengue. Thus, there is a strong need to develop therapeutic strategies that can tackle this life threatening disease. RNA interference is an important and effective gene silencing process which degrades targeted RNA by a sequence specific process. Several studies have been conducted during the last decade to evaluate the efficiency of siRNA in inhibiting dengue virus replication. This review summarizes siRNAs as a therapeutic approach against dengue virus serotypes and concludes that siRNAs against virus and host genes can be next generation treatment of dengue virus infection.

Nursing education has direct relationship with quality based clinical practice and standardized care; however, private versus public education makes a difference among the graduates. Unfortunately, in Pakistan, nursing education has been facing numerous challenges with respect of lack of unified policies, absence of strategic planning, and constraint resources. This paper describes comparison between private and public (government) nursing academic institution in Karachi, Pakistan. This paper comprises similarities, differences, strengths, limitations, and challenges of both institutions including their bases and recommendation for possible solution particularly with reference of regulatory body Pakistan Nursing Council (PNC) and World Health Organization (WHO) guidelines.

Background : Written Feedback (WFB) plays a significant role in student’s learning in terms of improvement of their academic writing skills. However, the quality of WFB may be affected by various personal and contextual factors. This study aimed to identify nurse teachers’ conceptions and practices of WFB on written assignments in nursing degree offering institutions in Karachi, Pakistan. Methods : A cross-sectional descriptive study design was used to answer the study questions. The study was conducted at 11 nursing schools in Karachi by recruiting those teachers who were teaching in nursing degree programs. The sample size of the study consists of 80 teachers. Universal sampling technique was used to select the subjects of the study. Results : The study result consists of demographic information of the participants, teachers’ conceptions and their practices of WFB. Although majority (92%) of the teachers believed that WFB is important for students’ learning, they acknowledged that they were not able to provide WFB at optimum level to their students. This gap in practice is attributed to various personal, contextual, and organizational factors; among them, the highest reported factors were teachers’ lack of training for provision of WFB (84%), distraction in the environment (78%), and constraints of time due to teacher’s workload (74%). Conclusions : The importance of WFB for students’ learning is well established in the literature. However, the preparation of teachers and an enabling environment to facilitate teachers for optimum practices of WFB needs considerable attention by educational institutions.

The purpose of this study was to characterize the five missense mutations in CYP1B1 gene identified in Pakistani families affected with primary congenital glaucoma (PCG) using various bioinformatics and protein modeling tools.We previously reported four novel missense mutations in CYP1B1 gene segregating in consanguineous Pakistani families. These mutations were identified by direct sequencing of all coding exons, the exon-intron boundaries and the 5' untranslated region of CYP1B1 using genomic DNA from affected and unaffected family members. In order to understand the effect of CYP1B1 mutations on protein structure and function, we used bioinformatics tools to investigate five mutations including four novels (W434R, D374E, L487P and L177R) and one known (E229K) mutation previously reported by our group in four Pakistani PCG-affected families.In silico analysis of the missense mutations using the computational algorithms SNAP, I-Mutant 2.0 IUPred, PrDOS and PASTA predicted pathogenic effects on stability and function of protein.In silico analysis of identified mutations confirmed their molecular pathogenicity. Similar analysis will be helpful in understanding of the biological role of CYP1B1 and the effect of mutations on the regulatory and enzymatic functions of CYP1B1 that result in PCG.

Many important cellular processes involve protein–protein interactions (PPIs) mediated by a Short Linear Motif (SLiM) in one protein interacting with a globular domain in another. Despite their significance, these domain-motif interactions (DMIs) are typically low affinity, which makes them challenging to identify by classical experimental approaches, such as affinity pulldown mass spectrometry (AP-MS) and yeast two-hybrid (Y2H). DMIs are generally underrepresented in PPI networks as a result. A number of computational methods now exist to predict SLiMs and/or DMIs from experimental interaction data but it is yet to be established how effective different PPI detection methods are for capturing these low affinity SLiM-mediated interactions. Here, we introduce a new computational pipeline (SLiM-Enrich) to assess how well a given source of PPI data captures DMIs and thus, by inference, how useful that data should be for SLiM discovery. SLiM-Enrich interrogates a PPI network for pairs of interacting proteins in which the first protein is known or predicted to interact with the second protein via a DMI. Permutation tests compare the number of known/predicted DMIs to the expected distribution if the two sets of proteins are randomly associated. This provides an estimate of DMI enrichment within the data and the false positive rate for individual DMIs. As a case study, we detect significant DMI enrichment in a high-throughput Y2H human PPI study. SLiM-Enrich analysis supports Y2H data as a source of DMIs and highlights the high false positive rates associated with naïve DMI prediction. SLiM-Enrich is available as an R Shiny app. The code is open source and available via a GNU GPL v3 license at: https://github.com/slimsuite/SLiMEnrich. A web server is available at: http://shiny.slimsuite.unsw.edu.au/SLiMEnrich/.

Fish is a diverse group of organisms living in different aquatic environment and containing almost all essential amino acids. Fourteen muscle proteins including titin, dystrophin, filamin, myosin heavy chain, spectrin, M1/M2, nebulin, alpha- actinin, gelsolin, actin, tropomyosin, troponin, thymosin and plastin3 were chosen for in-silico characterization. Sequence analyses were performed using BindN, Conseq, DIANNA, PROFEAT and ProtFun for exploiting structural and functional importance. Homology modeling technique was applied for predicting 3D structure which will assist in future for searching catalytic role of proteins in metabolic pathway. 3D Structure of eight muscle proteins was predicted using Protein Structure Prediction Server (PS 2 ) based on MODELLER algorithm. Phylogenetic relationship was inferred by sequence alignment through CLUSTAL X and furthermore phylogenetic tree was constructed by using MEGA which was statistically evaluated by DIVIEN. From structural analyses, these muscle proteins were inferred to contain functional domains, number of motifs, beta turns with important secondary structural features. Furthermore sequence study suggested, these proteins have important biochemical features such as number of cysteines, disulphide bonds, DNA and RNA binding sites, functionally conserved amino acid residues and were characterized as non-allergen proteins which can be used for designing effective vaccines. Overall, evidence from computational study revealed that these muscle proteins have structural and functional significance, which can play important role in drug designing and in exploring gene diversity. This novel approach to study muscle proteins would be beneficial for human since both vertebrates and invertebrates have muscle proteins in common.

With technological advancement, health care is becoming complex for the individuals and meeting individual expectations have become a great challenge for Health Care Providers (HCP). Health care providers face ethical dilemma in everyday situation for taking decision for patient’ better health outcomes. The aim of this paper is to highlight the controversy between giving patients’ autonomy in taking independent decisions and health care professionals’ expertise to breach patient autonomy in order to save a patient’s life and to provide maximum benefits to the patient. Moreover, this paper also highlights the significance of culturally sensitive care in health care setting. Research shows that in most of the situations, HCPs take decision in view of their expertise in the medical field. However, on many occasions, both HCP and patients are not satisfied with the decisions that are taken in the best interest of the patient.

Human immunodeficiency virus (HIV) significantly attributes to social stigmatization, besides being a health concern. It is one of the major responsibilities of the health care providers to practice caution before labeling a person with such diagnosis, as sometimes it is predicted to be false-positive. No doubt, health care employees are not exempted from acquiring the disease. However, inaccurate decisions could cause abundant consequences on a person’s personal and psychosocial wellbeing. Therefore, the higher management team of an entity has the prime responsibility to be fair with the employees and utilize the principle of justice in their actions. Indeed, moral responsibilities and ethical values play an important role in fairly decision-making in everyone’s life. Afghan women suffer from various unhealthy cultural and traditional practices besides other social inequalities. For this, strategies to encourage women in labor market and avoid gender discrimination must be launched. Furthermore, attempts to maintain justice through designing specific policy and protocol to avoid errors within an organization must be prioritized.

One of the important responsibilities of the health care system is to meet the health needs of the population mainly through health promotion and disease prevention initiatives. This objective can be achieved through an equitable resource distribution/allocation. This paper comprises of comparative analysis of health care system of two countries i.e., Pakistan and Japan in relation to the WHO recommended guidelines on Health care system. The analysis consists of organizational environment, health care delivery model, health information management system, health indicators, financing, and workforce of both the countries. This analysis suggests some strategies to overcome the shortcomings for both the countries. Link: http://aeirc-edu.com/ojs14/index.php/IJEHSR/article/view/163/261

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima β-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable β-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable β-glucosidase enzyme could be developed, enhancing its industrial potential.

Background: HCV has become a leading cause of liver cirrhosis and hepatocellular carcinoma and is a major health concern worldwide. To date, there is no vaccine available in the market to tackle this disease, therefore there is a strong need to develop antiviral compounds that can target all genotypes of HCV with the same efficiency. Medicinal plants have low cost and are less toxic therefore, extracts of medicinal plants can serve as important antiviral agents against HCV. This study was designed to screen phytochemicals of Accacia nilotica to find a potent drug candidate that can inhibit HCV infection effectively. Results: Docking of NS3/4A protease and Flavonoids of Accacia nilotica revealed that most of the flavonoids bound deeply with the active site of NS3/4A protease. Compound 01 showed a high ranking on docking score. All other compounds also showed reliable docking scores and had interactions with the binding cavity of NS3/4A protease, suggesting them as a potent drug candidate to block HCV replication. Conclusion: To recognize binding interactions of Accacia nilotica phytochemicals with NS3/4A protease, molecular docking was performed to find potential inhibitor against NS3/4A protease of HCV. After post docking analysis, important interactions were found between active compounds and active site of NS3/4A protease. It can be concluded from the study that phytochemicals of Accacia nilotica may serve as a potential drug candidate with relatively simple structural changes against HCV NS3/4A protease.

Background HCV is causing hundreds of cases yearly in Pakistan and has become a threat for Pakistani population. HCV E2 protein is a transmembrane protein involved in viral attachment and thus can serve as an important target for vaccine development but because of its variability, vaccine development against it has become a challenge. Therefore, this study was designed to isolate the HCV E2 gene from Pakistani HCV infected patients of 3a genotype, to perform In-silico analysis of HCV E2 isolated in Pakistan and to analyze HCV E2 protein sequence in comparison with other E2 proteins belonging to 3a and 1a genotypes to find potential conserved B-cells and T-cell epitopes that can be important in designing novel inhibitory compounds and peptide vaccine against genotype 3a and 1a. Patients and methods Patients were selected on the basis of elevated serum ALT and AST levels at least for six months, histological examination, and detection of serum HCV RNA anti-HCV antibodies (3rd generation ELISA). RNA isolation, cDNA synthesis, amplification, cloning and sequencing was performed from 4 patient’s serum samples in order to get the HCV E2 sequence. HCV E2 protein of Pakistani origin was analyzed using various bioinformatics tools including sequence and structure tools. Results HCV E1 protein modeling was performed with I-TASSER online server and quality of the model was assessed with ramchandran plot and Z-score. A total of 3 B-cell and 3 T-cell epitopes were found to be highly conserved among HCV 3a and 1a genotype. Conclusion The present study revealed potential conserved B-cell and T-cell epitopes of the HCV E2 protein along with 3D protein modeling. These conserved B-cell and T-cell epitopes can be helpful in developing effective vaccines against HCV and thus limiting threats of HCV infection in Pakistan.

Background HCV affects >170 million people worldwide and is a leading cause of liver diseases such as hepatocellular carcinoma. Each year, Pakistan reports hundreds of cases and now it has become a serious health issue. HCV has two transmembrane glycoproteins (E1 and E2) that are involved in virus entry through viral attachment, but because of their hypervariable nature they have become difficult targets for vaccine development. Methods A total of 150 protein sequences of E1 and E2 belonging to genotypes 3a and 1a were retrieved from the NCBI protein database and were subjected to conservation and variation analysis using the multiple sequence alignment feature of the CLC workbench. A consensus sequence of each genotype of E1 and E2 was obtained and these consensus sequences were further analyzed to construct a global consensus sequence, which was used to design potentially conserved peptides. Results From the sequence conservation analysis, highly conserved residues were identified and were used to design peptides. Only two peptides were found to be conserved in the E1 protein of genotypes 3a and 1a and a total of nine conserved peptides were designed for the HCV E2 protein of those genotypes. These designed peptides could serve as useful targets in developing new inhibitory compounds. Conclusion This study was designed to perform conservation and variability analysis of HCV glycoproteins and to find potentially conserved peptides among genotypes 3a and 1a (the most prevalent genotypes in Pakistan) that could serve as useful targets in the development of novel inhibitory compounds, thus reducing the threat of HCV infection in Pakistan.

HCV is a leading cause of liver disease and can lead to hepatocellular carcinoma and liver damage which in turn can cause death of patients. HCV is closely related to immune response especially inflammation suggesting that the immune response-related genes can serve as molecular targets for chemo-prevention and treatment of C-type HCC. This study was conducted to determine the 3D structures of immune responsive gene (CXCL6) that are up-regulated during Hepatitis C Virus (HCV) Infection and Hepatocellular Carcinoma (HCC) and HCV core protein.Furthermore, docking and interactions analysis of immune responsive gene with HCV core protein was performed to understand pathogenesis of HCV infection. Reliable 3D structures of both proteins were determined using comparative modeling approach. Docking of both proteins revealed functionally important residues i-e. Arg149, Arg39, Arg74 and Gln78 in HCV core protein and Leu44, Ala71, Ser76 and Pro97 in CXCL6. It can be concluded from the study that CXCL6 had potentially interacting residues with HCV core protein that can be helpful in finding clues to understand HCV pathogenesis and develop better therapeutic regimens.

Infection with Hepatitis C Virus (HCV) is a major health problem affecting 270 million people worldwide and 10 million people in Pakistan. Currently, there is no vaccine available for prevention of HCV infection due to the high degree of strain variation. The current standard of care is a combination of Pegylated interferon ? (PEG-INF ?) with ribavirin and Boceprevir/Telaprevir. Hence, there is a need to develop new antiviral agents from different targets. HCV NS3, a nonstructural protein has serine/protease and helicase domain, and these domains are considered as important antiviral drug targets to combat HCV. In the past 15 years, major scientific advances have enabled the development of many novel serine/protease and helicase inhibitors by using in vivo as well as in vitro model systems. Most of NS3 protease inhibitors have entered in different clinical trials phases but still there is a need to improve the functionality of these inhibitory compounds. This review summarizes potent antiviral compounds against HCV NS3 protease and their activity in combination with standard therapy. In conclusion, it will be very exciting to see HCV NS3-4A serine protease/helicase inhibitors progress through clinical developments and, hopefully, provide hepatitis C patients with much needed, more effective therapies.