Bacterial vaginosis: An insight into the prevalence, alternative treatments regimen and it's associated resistance patterns.

Bacterial Vaginosis (BV) is a complex polymicrobial infection of vagina that shifts the paradigms of vaginal flora from lactobacilli to opportunistic pathogens. BV is catagorized by greyish white discharge, pH greater than 4.5. It results in the preterm labor, abortion, pelvic inflammatory disorders, post cesarean infections. BV is associated with Sexually Transmitted Diseases (STDs) or immune deficiency disorders like Human Immunodeficiency Virus, Human Papilloma Virus, Herpes Simplex Virus 1 and 2, and Neisseria gonorrhoeae. The prevalence rate is about 21.2 million (29.2%) worldwide. BV is more frequent in black females as compared to white females, independent of geographical distribution. Globally, BV is treated with the current recommended antibiotic therapy including Metronidazole and Clindamycin. The recurrence rates are 76% and occur within 06 months of treatment due to antibiotic resistance against pathogenic bacteria and their biofilms. The antibiotic resistance is a global health issue which directs the attentions towards other treatments. One of these is the treatment of sex partners, thus helping to stop the recurrence rates in females. However, this method does not show any positive results. Probiotic therapy is an incorporation of Lactobacilli orally or intravaginally for the recolonization of healthy microbes. This therapy has exhibited promising results but some studies revealed that Probiotic therapy does not control the recurrence rate. The other methods are in trials period and none of them are used clinically or commercially available for the treatment. The thermoplastic polyurethane (TPU) intravaginal rings contain lactic acid and metronidazole showed promising results in trials of BV treatment. The vaginal acidifiers are used as an alternative method to maintain the vaginal pH but the process of douching is a major limitation. The activated charcoal is used to treat BV patients in clinical trials showed decrease in the pH with only 3.1% loss of lactobacilli. Phage therapy is a reemerging field to overcome the bacterial resistance. They are host specific and easier to handle. They can be used naturally, synthetically; phage cocktails and phage-antibiotics combination can be used. Phages show auspicious results for the treatment of bacterial infections as compared to antibiotics as they also treat biofilms. This is one of the promising therapy in future to treat infections with no side effects. Phage therapy can be used in pharmaceuticals according to Food and Drug Administration (FDA) guidelines. Taken together, it is suggested that large funding is required by pharmaceutical sector or government for further investigation of bacteriophages to be used against BV pathogenesis.

In-Vitro Transcription analysis of NS5A from HCV-3a circulating in Pakistani patients with chronic hepatitis C and their differential response to antiviral therapy.

OBJECTIVE: Mutations in HCV nonstructural protein 5A (NS5A) play a vital role in virus resistance. The aim of this study was to develop a correlation between NS5A mutations (genotype 3a) and virological response towards interferon alpha (IFN-α) plus ribavirin therapy. METHODS: In this study, which was conducted from 09-02-2013 to 25-11-2015 in the rural area of Province Sindh - Pakistan, total patients' responses to peg-IFN therapy were investigated. Patients were given peg-IFN therapy for 24 to 48 weeks and categorized as sustained virologic responders (SVR) or non-responders (NR) to HCV infection. HCV NS5A region (2215-2335) of genotype 3a was identified in both responders and non-responders. RESULTS: Twenty-four NR with 24 SVR isolates showed significant mutations within the nonstructural protein 5A region in HCV genotype 3a. The New Zealand (NZL1) (GenBank D17763) differences were observed by using gene. The ISDR mutations for nonstructural protein 5A in non-responders have been reported as a possible explanation of HCV interferon resistance. CONCLUSION: Based on these results, it is suggested that decreased SVR is caused by the increased mutations in nonstructural protein 5A sequences. When the sequence outside the Protein kinases R binding domain (PKRBD) (2281-2335) was examined, significant differentiations were observed among the SVR and NR classes at few amino acid strains.

Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome.

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

Current and future therapies for hepatitis C virus infection: from viral proteins to host targets.

Hepatitis C virus (HCV) infection is the most important problem across the world. It causes acute and chronic liver infection. Different approaches are in use to inhibit HCV infection, including small organic compounds, siRNA, shRNA and peptide inhibitors. This review article summarizes the current and future therapies for HCV infection. PubMed and Google Scholar were searched for articles published in English to give an insight into the current inhibitors against this life-threatening virus. HCV NS3/4A protease inhibitors and nucleoside/nucleotide inhibitors of NS5B polymerase are presently in the most progressive stage of clinical development, but they are linked with the development of resistance and viral breakthrough. Boceprevir and telaprevir are the two most important protease inhibitors that have been approved recently for the treatment of HCV infection. These two drugs are now the part of standard-of-care treatment (SOC). There are also many other drugs in phase III of clinical development. When exploring the various host-cell-targeting compounds, the most hopeful results have been demonstrated by cyclophilin inhibitors. The current SOC treatment of HCV infection is Peg-interferon, ribavirin and protease inhibitors (boceprevir or telaprevir). The future treatment of this life-threatening disease must involve combinations of therapies hitting multiple targets of HCV and host factors. It is strongly expected that the near future, treatment of HCV infection will be a combination of direct-acting agents (DAA) without the involvement of interferon to eliminate its side effects.

Green synthesised AuNps using Ajuga Bracteosa extract and AuNps-Free supernatant exhibited equivalent antibacterial and anticancerous efficacies.

The current study is designed to synthesize gold nanoparticles using Ajuga bracteosa extract, which is a highly known medicinal herb found in the northern Himalayas. The synthesized gold nanoparticles were initially characterized by UV-Vis spectrophotometer, SEM, FTIR, pXRD, and, GC-MS. Antibacterial efficacy of A. bracteosa extract, AuNps, and AuNps-free supernatant activity was checked against highly pathogenic clinical isolates of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa via agar well diffusion method, assuming that supernatant might have active compounds. The Nps-free supernatant showed the maximum antibacterial activity against E. coli (20.8±0.3 mm), Staphylococcus aureus (16.5±0.5), and Pseudomonas aeruginosa (13±0.6). While green synthesized AuNps showed effective antibacterial activity (Escherichia coli (16.4±0.3mm), Staphylococcus aureus (15.05±0.5mm), and Pseudomonas aeruginosa (11.07±0.6mm)) which was high compared to A. bracteosa extract. Anticancer activity was assessed by MTT assay on U87 and HEK293 cell lines. Aj-AuNps have an antigrowth effect on both the cell lines however Aj-AuNps-free supernatant which was also evaluated along with the Aj-AuNps, showed high toxicity toward HEK293 cell line compared to U87. Further, the GC-MS analysis of supernatant showed the presence of resultant toxic compounds after the reduction of gold salt, which include Trichloromethane, Propanoic acid, 2-methyl-, methyl ester, Methyl isovalerate, Pentanoic acid, 2-hydroxy-4-methyl-, Benzene-propanoic acid, and alpha-hydroxy. Based on the observation small molecular weight ligands of Ajuga bracteosa were analyzed in-silico for their binding efficacy towards selected membrane proteins of our target pathogens. RMSD is also calculated for the best docked protein ligand pose. The results revealed that among all listed ligands, Ergosterol and Decacetylajugrin IV have high virtuous binding affinities towards the membrane proteins of targeted pathogens. The current findings revealed that the Aj-AuNps are good antibacterial as well as anticancerous agents while the Nps-free supernatant is also exceedingly effective against resistant pathogens and cancer cell lines.

Computational design of experimentally validated multi-epitopes vaccine against hepatitis E virus: An immunological approach.

Hepatitis E virus (HEV) is one of the leading acute liver infections triggered by viral hepatitis. Patients infected with HEV usually recover and the annual death rate is negligible. Currently, there is no HEV licensed vaccine available globally. This study was carried out to design a multi-epitope HEV peptide-based vaccine by retrieving already experimentally validated epitopes from ViPR database leading to epitope prioritization. Epitopes selected as potential vaccine candidates were non-allergen, immunogenic, soluble, non-toxic and IFN gamma positive. The epitopes were linked together by AAY linkers and the linker EAAAK was used to join adjuvant with epitopes. Toll-like receptor (TLR)-4 agonist was used as an adjuvant to boost efficacy of the vaccine. Furthermore, codon optimization followed by disulfide engineering was performed to analyse the designed vaccine's structural stability. Computational modeling of the immune simulation was done to examine the immune response against the vaccine. The designed vaccine construct was docked with TLR-3 receptor for their interactions and then subjected to molecular dynamic simulations. The vaccine model was examined computationally towards the capability of inducing immune responses which showed the induction of both humoral and cell mediated immunity. Taken together, our study suggests an In-silico designed HEV based multi-epitope peptide-based vaccine (MEPV) that needs to be examined in the wet lab-based data that can help to develop a potential vaccine against HEV.

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits.

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019-2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96-99.5% nucleotide and 94.40-99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li's F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.

Hepatitis C virus infection: molecular pathways to insulin resistance.

Chronic Hepatitis C virus has the potential of inducing insulin resistance and type 2 Diabetes Mellitus in vitro as well as in vivo . Structural and non-structural proteins of HCV modulate cellular gene expression in such a way that insulin signaling is hampered, concomitantly leads toward diabetes mellitus. A number of mechanisms have been proposed in regard to the HCV induced insulin resistance involving the upregulation of Inflammatory cytokine TNF-α, hypophosphorylation of IRS-1 and IRS-2, phosphorylation of Akt, up-regulation of gluconeogenic genes, accumulation of lipids and targeting lipid storage organelles. This review provides an insight of molecular mechanisms by which HCV structural and non-structural proteins can induce insulin resistance.

Multifaceted role of cancer educated platelets in survival of cancer cells.

Platelets, the derivatives of megakaryocytes, pose dynamic biological functions such as homeostasis and wound healing. The mechanisms involved in these processes are utilized by cancerous cells for proliferation and metastasis. Platelets through their activation establish an aggregate termed as Tumor cell induced platelet aggregation (TCIPA) that aids in establishing a niche for the primary tumor at secondary site while recruiting granulocytes and monocytes. The study of these close interactions between the tumor and the platelets can be exploited as biomarkers in liquid biopsy for early cancer detection, thereby increasing the life expectancy of cancer patients.

Physicochemical, Sensorial and Microbiological Characterization of PoroCheese, an Artisanal Mexican Cheese Made from Raw Milk.

Poro cheese is a regional product originally from the area of Los Rios, Tabasco in Mexico. In the context of preserving the heritage of Poro cheese and protecting the specific characteristics that define its typicity through an origin designation, the present study was conducted to establish a general profile of Poro cheese by characterizing their physicochemical, textural, rheological, sensorial and microbiological characteristics. Differences in moisture, proteins, fats, NaCl, titrable acidity, pH, color texture and rheology amongst cheese factories were observed and ranges were established. Fifteen descriptors were generated to provide a descriptive analysis, eight of which were significantly different amongst the factories with no differences in the global acceptability of cheese. The favorite cheese had the highest scores for aroma attributes. Conventional and molecular methods were used to identify the main microorganisms, for which Lactobacillus plantarum, L. fermentum, L. farciminis and L. rhamnosus were the main microorganisms found in Porocheese. The obtained data constituted the parameters for characterizing Poro cheese, which will strongly help to support its origin appellation request process.

In silico analysis of glycinamide ribonucleotide transformylase inhibition by PY873, PY899 and DIA.

In humans, purine de novo synthesis pathway consists of multi-functional enzymes. Nucleotide metabolism enzymes are potential drug targets for treating cancer and autoimmune diseases. Glycinamide ribonucleotide transformylase (GART) is one of the most important trifunctional enzymes involved in purine synthesis. Previous studies have demonstrated the role of folate inhibitors against tumor activity. In this present study, three components of GART enzyme were targeted as receptor dataset and in silico analysis was carried out with folate ligand dataset. To accomplish the task, Autodock 4.2 was used for determining the docking compatibilities of ligand and receptor dataset. Taken together, it has been suggested that folate ligands could be potentially used as inhibitors of GART.

Ionotropic Purinergic Receptors P2X4 and P2X7: Proviral or Antiviral? An Insight into P2X Receptor Signaling and Hepatitis C Virus Infection.

Purinergic P2X receptors are plasma membrane bound, ATP-gated ion channels that are expressed on wide range of cells and respond to varying ATP concentrations in extracellular environment. Upon activation they increase membrane permeability for Ca(2+) ions and trigger a cascade of signaling complexes. During the course of hepatitis C virus (HCV) infection, ATP is released from the infected hepatocyte, which binds with Purinergic receptors (P2X) on peripheral blood mononuclear cells (PBMCs) and initiate downstream signaling pathways by disturbing the ionic balance of the cell. The present study investigates quantitative expression of P2X7 and P2X4 along with selected host genes PEPCK, transforming growth factor ß (TGF-ß), MAPK, Rho, and Akt in PBMCs of chronic HCV infection patients. PBMCs were isolated from collected blood samples of study subjects. Transcript analysis of P2X7, P2X4, and targeted downstream genes was done using quantitative real-time polymerase chain reaction. Relative expression analysis was performed by unpaired Student's t test on GraphPad Prism version 5. We found a notable increase of threefolds and 1.8-folds in the expression of P2X7 and P2X4 receptors in treatment naïve category while the expression of PEPCK, TGF-ß, MAPK, AKT, and Rho A increased by 2.8, 1.9, 2.2, 2.2, and 1.8-folds, respectively. In sustained virological response patients, P2X7 significantly increased up to 3.5-folds while the expression of P2X4 receptor was increased up to twofold. In third category, treatment nonresponder, the expression of P2X7, P2X4 receptors, and targeted markers remained un-altered. This study deals with two major aspects of P2X4 and P2X7 receptors in PBMCs of chronic HCV individuals. One is their role in providing antiviral immunity to host against HCV; second aspect is the role of P2X receptors in inducing HCV pathogenesis via AKT, TGF-ß, Rho A, PEPCK, and MAPK expression.

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis.

AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus (HCV) nonstructural protein 5A (NS5A). METHODS: The human hepatoma cell lines, Huh7 and Huh7.5, were infected with HCV or transiently-transfected with a vector expressing HCV NS5A. The effect of HCV NS5A on the status of the critical players involved in insulin signaling was analyzed using real-time quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0. RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway, we analyzed the status of insulin receptor substrate-1 (IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1 (Ser(307)) in HCV infected or NS5A transfected Huh7.5 cells compared to their respective controls. Furthermore, an increased phosphorylation of Akt (Ser(473)) was observed in HCV infected and NS5A transfected cells compared to their mock infected cells. In contrast, we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue (Ser(307)) followed by decreased phosphorylation of Akt Thr(308), Fox01 Ser(256) and GSK3ß Ser(9), the downstream players of the insulin signaling pathway. Furthermore, increased expression of PECK and glucose-6-phosphatase, the molecules involved in gluconeogenesis, in HCV NS5A transfected cells was observed. CONCLUSION: Taken together, our results suggest the role of HCV NS5A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.

Predictive potential of IL-18 -607 and osteopontin -442 polymorphism in interferon-based therapy of HCV infection in the Pakistani population.

The adaptive immune system plays an important role in response to interferon plus ribavirin treatment of hepatitis C virus (HCV) infection. Cytokines play a significant role in the adaptive immune system. The production of cytokines may be regulated by single nucleotide polymorphisms (SNPs). This study was designed to examine the correlation of some important SNPs of cytokines with interferon plus ribavirin treatment of HCV infection in the Pakistani population. We followed 140 chronic HCV-infected patients in our study. All of these patients had completed their planned course of interferon plus ribavirin treatment. We also considered 120 healthy subjects as controls. The detection of interleukin-18 (IL-18) SNPs was performed by tetra-primers amplification-refectory mutation system polymerase chain reaction, while for genotyping of osteopontin (OPN), transforming growth factor beta (TGFß), and N-acetylgalactosaminyltransferase 8 (GALNT8) SNPs, allele-specific polymerase chain reaction was performed. The distribution of the IL-18 -607AA genotype varied significantly between healthy control and patient groups. Its distribution was significantly high in healthy subjects than HCV patients (p = 0.031), signifying its potential involvement in the natural clearance of HCV infection. The occurrence of the -607AA genotype of IL-18 was also significantly higher in the sustained virological group (SVR) than in the nonresponder (NR) group (p = 0.046), highlighting its protective involvement in the treatment outcome of chronic HCV infection. The frequency of the OPN -442TT genotype was higher in the SVR group than in the NR group (p = 0.034), indicating a significant possible role of this genotype in therapy for HCV infection. No important association was found between TGFß and GALNT8 genotypes and the natural clearance and treatment response of HCV infection. IL-18 -607AA and OPN -442TT genotypes can be used as positive predictive markers of interferon plus ribavirin treatment of HCV infection in the Pakistani population.

Development of murine models to study Hepatitis C virus induced liver pathogenesis.

Hepatitis C virus (HCV) is involved in different liver pathologies worldwide. In contemporary scenario, HCV treatment is lagging behind owing to absence of vaccines against virus. The only consideration for HCV treatment is pegylated interferon-alpha and ribavirin that results in sustained virological response in 50 % of patients. Two feasible hosts for HCV infection are chimpanzee and humans. For decades, chimpanzees are sole host to study HCV pathogenesis, but their use is limited due to ethical issues. The dilemma behind HCV therapy is the need of sustainable animal models that can help simulate in vivo conditions. We have assembled recent advances in animal models to study liver diseases for targeted therapy.

An overview: in vitro models of HCV replication in different cell cultures.

Although much of productive research has been conducted in the field of molecular virology of Hepatitis C virus (HCV) regarding its genes, gene functions and proteins, development of an efficient cell culture model for its replication remained a focused area. Focus has been directed to establish HCV in vitro replication system. This replication system should mimic its intrahepatic pathogenesis so that antivirals should be screened and in vitro gene profiling of HCV induced pathogenesis should be worked out. Since 1990 various experimental approaches and strategies have been utilized in phase of development of a robust replication model for HCV, and success has been reported for a few genotypes. Still the work is going on to have more success in availing such robust replication models for all the genotypes. This will help to have a common antiviral strategy against HCV induced pathogenesis involving any genotype or subtype.