Evaluation of autophagy induction and inhibition in the Huh7.5 cell line through flow cytometry

Background: Autophagy is a physiologic process in which double membrane vesicles engulf damaged proteins and organelles for delivering them to lysosomein order to degrade and recycle them via lysosomal digestion. Beclin1 is one of the basic proteins involved in the initial step of autophagosome formation. In the current study, the effect of exogenous Beclin1 to induce autophagy and the effect of 3MA to inhibit of autophagy was assessed in Huh7.5 cells as an in vitro models of hepatocellular carcinoma. Material and methods: The Recombinant pcDNA-Beclin1was transfected into Huh7.5 cells. Also, the cell treated with 3MA. Next, the autophagy induction and inhibition was conducted via LC3 staining as a main autophagy marker using flow cytometry. Results: The result of this study suggest that the over expression of exogenous Beclin1 in Huh7.5 cells elevated the autophagosome formation as shown by intracellular autophagosomal marker LC3-II staining for about 32.32 % and   3MA decreased  it up to2% in compared with control cells in which the stained LC3-II was12.08. Conclusion: Recombinant beclin1 may be used as a potential autophagy inducer agent and 3-methyl-Adenin inhibits autophagy formation in Huh7.5 cell. The staining autophagy formation marker LC3-II with specific antibody is a reliable method to measure autophagy activation via flow cytometry

Determining the Demographic and Histopathological Pattern of Basal Cell Carcinoma in the Pathology Laboratories of Babol University of Medical Sciences from 2013 to 2020

Background and Objective: Basal cell carcinoma is the most common human malignancy that has been increasing in recent years. The aim of this study is to determine the demographic and histopathological pattern of this malignancy in the pathology departments of state hospitals in Babol from 2013 to 2020. Methods: In this retrospective study, after reviewing the existing files with the final diagnosis of basal cell carcinoma from 2013 to 2020, the patients' information including age, gender, lesion location and microscopic subgroup were recorded and analyzed. Findings: In this study, 367 cases were obtained from Shahid Beheshti and Shahid Yahyanejad Hospitals in Babol with the diagnosis of basal cell carcinoma. The mean age of people in both genders was 67±10.89 years. 69% of the samples were men and 31% were women (p<0.001). The highest frequency was from the scalp area (134 samples) and the lowest frequency was related to the chest (3 samples). The most common histopathology subtype was nodular type and the rarest types were metatypical and clear cell carcinoma. A statistically significant relationship was observed between the location of the sample and gender (p=0.023); the cheek in men and the nose in women were more common than the other gender. The highest incidence was seen in 2017 with 83 cases, which was a significant increase compared to the previous year. Conclusion: Based on the results of this study, the high incidence of basal cell carcinoma in the head and face area of elderly people, especially men, reveals the need to inform high-risk communities and to be more familiar with therapists, especially dentists who are in close eye contact with the face

What is the best laboratory method for diagnosis of Herpes Simplex Virus in genital infections?

  Genital infection caused by Herpes simplex virus (HSV) is one of the most common health problems, worldwide. Several methods such as cell culture, serological and molecular methods have been used to detect this virus. Currently, Real-Time Polymerase Chain Reaction (Real time-PCR) technique is widely used due to its high sensitivity and specificity. Besides, Real time-PCR can be employed in the follow-up of therapeutic effects in HSV-infected person who is being treated with antiretroviral drugs. We conducted a review on traditional and current diagnostic methods with a focus on their limitations in the diagnosis of HSV infection

Design, construction and evaluation of 1a/JFH1 HCV chimera by replacing the intergenotypic variable region

Peer reviewedPublisher PD

Epidemiology and Clinical Characteristics of Patients with Hepatocellular Carcinoma in North-East of Iran

Background: Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a life-threatening disease worldwide. The aim of this study was to investigate the epidemiology and clinical features of HCC patients who referred to Omid hospital in Mashhad, northeast of Iran.Materials and Methods: In this cross sectional retrospective study, we reviewed the medical records of patients who referred to Omid hospital – a cancer research center– in Mashhad during 1991 to 2012. Medical records of 29 patients with primary liver cancer proven with biopsy, CT scan or MRI were analyzed in this study.Results: Of 25 eligible cases, 68% were men and the rest were women. The majority of HCC patients were in the 60-69 age group. Also, 44% of patients were found to be hepatitis B virus surface antigen (HBsAg) positive.Conclusion: The age distribution and male preponderance of HCC patients observed in the present study in line with other conducted studies in Iran and other countries. Since this is a retrospective study, a comprehensive study with a larger sample size in a case-control study is needed to establish other HCC-related factors in our province

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle

Functional interactions of structural and NS proteins of hepatitis c virus

Hepatitis C virus (HCV) is a small enveloped virus with a positive-sense single stranded RNA genome. Based on its molecular genetic characteristics, the virus has been classified into the Hepacivirus genus of the family Flaviviridae. HCV is one of the major causes of chronic hepatitis which can lead to liver cirrhosis and hepatocellular carcinoma. According to the recent WHO published data, 123 million individuals are infected with HCV (approximately 3% of the world’s population) throughout the world. Due to its highly variable nature, HCV is classified into six major genotypes. The HCV genome encodes a single polyprotein that is cleaved to yield at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The recently developed HCV cell culture system, based on the JFH1 strain of HCV, has provided an opportunity to study the role of the viral proteins in the complete HCV replication cycle in human hepatoma cells. How the viral proteins functionally interact during replication of HCV in cell culture is not completely understood. Passage of cell cultures transfected with HCV genomic RNA containing attenuating mutations allows for the selection of genomes with second site compensatory mutations that restore replication to wild type levels. Using this approach, the functional interactions of p7 and E2 with other viral proteins during HCV replication was investigated. A small protein of 63 amino acids, p7 is encoded at the junction of the structural and non-strucutural region. p7 is a highly hydrophobic, integral membrane protein and is classified in the viroporin family. In this thesis, it is shown that p7 is critical for production of viral particles and is implicated in a late step of particle assembly. Since the protein plays a critical role in the virus life cycle, chemical compounds that block p7 function are potential candidates for anti-viral therapy. In this thesis, a chimeric JFH1 genome that encodes the p7 protein of genotype (GT) 1b strain J4 was generated. The intergenotypic chimeric genome was nonviable in human hepatoma cells and infectious chimeric virions were only produced after cells harboring the chimeric genomes were passaged several times. To investigate the emergence of compensatory mutations in the viral proteins during cell passaging, the consensus sequences of the entire polyprotein coding regions of the wild type JFH1 and three chimeric viruses were determined. Sequence analysis revealed mutations in core, NS2, NS5A and NS5B. Reverse genetic analysis demonstrated that any one of the single mutations restored the infectivity of the defective chimeric genomes. These data suggest that there are critical genetic interactions between p7 with core, NS2, NS5A and NS5B. In addition, a stable physical interaction between p7 and NS2 is shown in a transient expression system. The HCV glycoproteins E1 and E2 are present on the surface of virions as a heterodimer that attach virions to host cell receptors and facilitate virus fusion and entry. HCV entry proceeds via attachment to glycosaminoglycans followed by binding to scavenger receptor type B class I, and the tetraspanin CD81. Recently, claudin-1 and occludin have emerged as additional receptors required for entry. E2 has a receptor binding domain (E2661RBD) that conatins three variable regions, hypervariable regions 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). In this thesis, HVR1 of E2 was deleted in the context of full-length replication comptetent HCV. Deletion of HVR1 increases CD81-binding ability of the mutant and also increases its susceptibility to neutralizing antibody MAb 24. The infectivity of the HVR1 deleted virions was attenuated approximately 10-fold prior to accumulation of compensatory mutations. Sequencing of cDNA obtained from reverted virions revealed mutations in E1 (I262L) and E2 (N415D). Reverse genetic studies revealed that I262L improved the infectivity of HVR1 deleted virions 2.5 fold while N415D restored infectivity to wild type levels. These data suggest that mutations within E1 or E2 can compensate for the reduction in infectivity observed for HVR1 deleted viruses. In summary, this thesis demonstrates the importance of functional interactions between HCV proteins during virus morphogenesis and infectivity

Prevalence and Characteristics of Precore Mutation in Iran and Its Correlation with Genotypes of Hepatitis B

Introduction: Mutation of the HBV precore gene prevents the production of HBeAg, which is an important target for immune responses. Distribution of this mutation varies along with frequency of HBV genotypes in accordance with geographic and ethnic variations. The general objective of this study was to evaluate the prevalence and characteristics of precore mutation in Iran and its correlation with genotypes of hepatitis B. Methods: In this cross-sectional study, viral DNA of 182 Iranian hepatitis B surface antigen positive patients who were admitted to Bandar Abbas Blood Transfusion Organization in 2012 and 2013 was retrieved from their serum samples. HBeAg, anti-HBe, and anti-HBc IgM diagnostic tests were performed using ELISA kits. Precore and Pre-S regions were amplified using specific primers and PCR thereafter to determine the genotypes; precore mutation, PCR, and restriction fragment length polymorphism (RFLP) methods also were applied. SPSS version 12 was used for data analysis by Mann–Whitney U test, Fisher’s exact probability test, and t-test. Results: A total of 62 patients (34.1%) had precore mutation (A1896G), and genotype D was the predominant genotype in these patients, which was followed by an unknown genotype that was suspected for genotype B. Interestingly, the relationships between precore mutation and HBeAg (p=0.037) and genotype D (p=0.005) were significant; however, no correlation was observed between this mutation and acute or chronic hepatitis and sex of patients. Conclusion: This study found high prevalence of precore mutations in southern Iran, which was significantly associated with HBeAg and genotype D

Application of bacterial shuttle vectors in designing new vaccines against infectious diseases: brief report

Background: Today, several vaccines have been developed to prevent infectious diseases. The older first-generation vaccines may have many problems. In this regard, genetic engineering plays an important role using tools such as shuttle vectors to develop recombinant DNA vaccines that usually include plasmid constructed so that can propagate in two different host species. The present study reviews a variety of shuttle vectors, their structures, productions, pathogenicity and more importantly their applications in the production of novel vaccines. Methods: A systematic review was performed based on search in international databases with no time limit including Scopus, PubMed and Google Scholar. All databases were searched using the standard (English and Persian) keywords. Relevant articles from 1996 to 2018 were collected from search of international databases including Science Direct, Google Scholar, and PubMed using keywords such as “shuttle vectors”, “recombinant plasmids” and “DNA vaccines”. Results: In this study, a total of 31 full texts were used. A shuttle vector typically contains similar components to replication origins and promoters and can propagate in various hosts. Nowadays, they are used in designing and constructing of new vaccines against infectious diseases including tuberculosis and viral hepatitis. Also, Multi-epitope peptide DNA vaccines are effective against some viruses and they are potentially effective against some bacteria such as Helicobacter pylori. Conclusion: Shuttle vectors as a powerful genetic engineering tool have a high ability to study the mechanisms of pathogenic microorganisms and make new vaccines such as DNA vaccines and multi-epitope vaccines. The hope is that such multi-epitope DNA vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. However, the ability of DNA vaccination to co-deliver a series of antibody and/or CD4 T cell epitopes remains largely unexplored