Eukaryotic expression of the core gene of hepatitis C virus genotype 1a

Background: Worldwide, hepatitis C virus (HCV) infection is a serious public health disease unlike hepatitis A and B, there is currently no vaccine against HCV available. Thus, extensive studies are under way to design new and effective treatments against HCV. Core protein is a component of HCV particle which is the first antigen recognized by the immune system.beside protective properties of core protein, anti –core antibodies can be used to monitor the disease progress. The purpose of the present study was to isolate and clone the core (C) gene from HCV genotype 1a in an attempt to construct a recombinant vector and subsequently evaluate its expression in a cell culture system.Methods:RNA genome of HCV genotype 1a was extracted from the blood of an infected patient. Complementary DNA (cDNA) was synthesized. HCV 1a core gene was amplified by PCR using specific primers and it was cloned into a eukaryotic expression vector. Huh7.5 cells were transfected by the designed recombinant vector and the cellular expression of the core gene was confirmed by RT-PCR.Results: Recombinant pcDNA3.1 (+) vector containing the HCV core gene with approximate size of 576bp was successfully designed. RT-PCR was used to confirm the expression of core antigen in an Huh7.5 cell line.Conclusion: The results showed that the core gene was successfully isolated from HCV genotype 1a and was cloned into the eukaryotic expression vector. This recombinant vector effectively replicated in Huh7.5 cell line. and its protective and therapeutic effects can be examined in further investigations

Prevalence of chromosomal aberrations in couples with recurrent miscarriages in the city of Mashhad, Iran: a cross-sectional study

Background: Recurrent miscarriage is defined as two or more recurrent spontaneous miscarriages. Several causes have been suggested, among which, chromosomal abnormalities in couples is considered to have a role in this regard. However, its significance varies among different populations. The present study was carried out to evaluate the prevalence of chromosomal aberrations in couples with recurrent miscarriages in the city of Mashhad.Materials and Methods: A retrospective study was performed on patient records at Medical Genetics Clinic of Imam Reza hospital in Mashhad (north-east of Iran) between 2003 and 2006.Results: Of 151 records of recurrent miscarriages, 59 couples had undergone Karyotyping testing. Among those who had Karyotyping results, only one (1.7%) had chromosomal abnormality. The observed abnormality was associated with chromosome 9 inversion. The prevalence of consanguineous marriage among these couples was 59.0%.Conclusion: In our study, unlike similar studies in other countries, the prevalence of chromosomal abnormalities was much lower. This could be interpreted either due to laboratory errors in our clinic or the real reduction in the association of chromosomal abnormalities with recurrent miscarriages in our population. Regarding our data, it seems that, at least among our population, costly Karyotyping testing is not necessary to predict further miscarriages or it could be limited to fewer cases having other associated factors

Comparison of the Prevalence of Human Papillomaviruses among Fertile and Infertile Women in Mashhad, Northeast of Iran

Background: Human papillomaviruses (HPVs) are the most common viruses which can be sexually transmitted. They can cause different malignancies in asymptomatic women. The association of HPVs with infertility among men and women is controversial. In the current study, the authors compared the frequency of HPVs in fertile and infertile women in the city of Mashhad. Materials and Methods: In the present case-control study, cervical and vaginal smears were collected from infertile and fertile women. HPVs were detected by polymerase chain reaction. Data was analyzed by SPSS v.20 and P-value <0.05 was considered statistically significant. Results: In the current study, 115 infertile women with the mean age of 30.5±5.6 years and 60 fertile women with the mean age of 32.6±9.3 years were included (p=0.07). Among women who were infertile (cases), 121 (52.6%) of 230 smears were positive, while in control group (who were fertile), 50 (41.7%) of 120 smears were positive (p=0.052). Conclusion: Frequency of HPV in both groups was high, which could be due to lack of routine HPV vaccination. HPV can cause placenta abnormality, our infertile women had multiple abortion history and history of abortion had significant differences among infertile and control group. The frequency of HPV had no significant differences between the infertile and control groups

Designing and construction a DNA vaccine encoding the fusion fragment of cfp10 and Ag85A immunodominant genes of Mycobacterium tuberculosis

Background: Pathogenic mycobacteria are one of major causes of human morbidity and mortality. Mycobacterium tuberculosis (M. tuberculosis) is an etiological agent of human tuberculosis. Designing new vaccines including DNA vaccines may be considered as new approaches for preventing of TB.Materials and Methods: M. tuberculosis H37Rv was grown on Lowenstein Jensen medium for 4 weeks at 37ºC and then DNA was extracted. The cfp10 gene was amplified by PCR. After digesting the PCR product and the plasmid, cfp10 fragment was ligated into the vector using T4 DNA ligase. Then, Ag85A was subcloned into pcDNA/cfp10. Escherichia coli strain JM109 bacteria were transformed by the desired construct. Clone confirmations were performed by colony PCR, restriction enzyme digestion and DNA sequencing. Recombinant vector was transfected into HeLa cells and total RNA was extracted, then cDNA was synthesized using oligo-dT. Finally PCR was performed by cfp10 primers.Results: The cfp10 was amplified by PCR method and the PCR products were visualized by agarose gel electrophoresis. The cfp10 fragments showed 303 bp in length. The cfp10 cloned into pcDNA. Then, Ag85Awas ligated into pcDNA/cfp10 after digestion correctly. Colony-PCR and restriction enzyme digestion and sequencing confirmed the cloning the fusion Ag85A/cfp10 fragment. Finally, after cDNA synthesis, expression of vector was confirmed in eukaryotic system.Conclusion: Cloning of Ag85A/cfp10 genes of M. tuberculosis were performed correctly. It can use as a DNA vaccine for investigation the immune responses in animal models in future studies

Isolation, cloning and molecular analysis of ag85a and tb10.4 genes from Mycobacterium tuberculosis

Background: Novel tuberculosis (TB) vaccines that aim to boost and/or replace Bacillus Calmette-Guerin (BCG) are currently in development. DNA vaccines can stimulate both humoral and cell-mediated immunity in different animal models of TB and is thought to be a promising strategy in the development of new vaccines against TB. The aim of this study was to design and construct a DNA vaccine encoding ag85a and tb10.4 fusion genes of Mycobacterium tuberculosis.Materials and Methods: tb10.4 fragment was amplified by PCR and the product was digested with restrictionenzymes. Next, it was cloned into the pcDNA3.1+ plasmid. The ag85a gene and pcDNA3.1+/tb10.4 plasmid were digested by EcoRI and BamHI restriction enzymes. Constructed vector was sequenced. The molecular analysis was done using bioinformatics software. New chimeric vector containing ag85a-tb10.4 genes were purified. Expression of pcDNA3.1+/tb10.4-ag85a plasmid was confirmed in eukaryotic cells.Results: Fragments of 297 bp for tb10.4 and 1017 bp for ag85a were observed in agarose gel electrophoresis.Alignment of ag85a-tb10.4 genome sequence with reference genes in GenBank showed exact identities that indicate correction of all cloning procedures. Transfection of eukaryotic cells with pcDNA3.1+/tb10.4-ag85a vector and existence of tb10.4-ag85a fusion gene were both confirmed with RT-PCR.Conclusion: In this study, tb10.4 and ag85a genes were isolated from Mycobacterium tuberculosis H37Rv strain and cloned into pcDNA3.1+. Also, the capability of constructed vector in producing fusion ag85a-tb10.4 protein was confirmed with RT-PCR. pcDNA3.1+/tb10.4-ag85a vector can be used for further studies in future

No Evidence of Hepatitis C Virus Infection in Individuals with Cardiovascular Disease in Mashhad

Background and Aim: Hepatitis C virus (HCV) infection is one of the leading causes of morbidity and mortality worldwide. It has been hypothesized that a number of bacteria and viruses might be involved in the pathogenesis of cardiovascular disease. The aim of this study was to define the prevalence of HCV in patients with cardiovascular disease in comparison with a control group. Methods: In this study, 281 individuals including 143 cardiovascular patients and 138 healthy controls were assessed for identification of HCV antibodies. The data collection was done between April 2016 and February 2017. The prevalence of HCV antibodies was determined by the enzyme-linked immunosorbent assay (ELISA) method. Results: There was no HCV infection in both patients with or without cardiovascular disease. There was a significant direct correlation between cardiovascular diseases and mean level of FPG (Fasting plasma glucose) (p<0.001). Also the Systolic and Diastolic blood pressures were significantly higher in the patients with cardiovascular disease (p<0.001 and p=0.005, respectively). Conclusion: The results of this study show that no evidence of HCV infection is found among a group of cardiovascular patients in the city of Mashhad. *Corresponding Author: Zahra Meshkat; Email: [email protected] Please cite this article as: Shakeri Hoseinabad M, Aryan E, Ghayour Mobarhan M, Moohebati M, Abolbashari S, Gholoobi A, Houshyar Chechaklou A, Yaghoubi A, Meshkat M, Meshkat Z. No Evidence of Hepatitis C Virus Infection in Individuals with Cardiovascular Disease in Mashhad. Arch Med Lab Sci. 2021;7:1-5 (e13). https://doi.org/10.22037/amls.v7.3344

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

Macular Amyloidosis and Epstein-Barr Virus

Background. Amyloidosis is extracellular precipitation of eosinophilic hyaline material of self-origin with special staining features and fibrillar ultrastructure. Macular amyloidosis is limited to the skin, and several factors have been proposed for its pathogenesis. Detection of Epstein-Barr virus (EBV) DNA in this lesion suggests that this virus can play a role in pathogenesis of this disease. Objective. EBV DNA detection was done on 30 skin samples with a diagnosis of macular amyloidosis and 31 healthy skin samples in the margin of removed melanocytic nevi by using PCR. Results. In patients positive for beta-globin gene in PCR, BLLF1 gene of EBV virus was positive in 23 patients (8 patients in case and 15 patients in the control group). There was no significant difference in presence of EBV DNA between macular amyloidosis (3.8%) and control (23.8%) groups (P=0.08). Conclusion. The findings of this study showed that EBV is not involved in pathogenesis of macular amyloidosis

Comparing the adverse outcomes of contraception failure between IUD and withdrawal methods

Background: Objective of current study was to compare the adverse outcomes of pregnancy after failure of IUD (Intrauterine device) with the withdrawal method of contraception in order to predict and prevent such outcomes.Methods: In a retrospective cohort study, the adverse outcomes of 224 pregnancies (2 groups, 112 women each) were assessed following failure of the IUD or withdrawal methods of contraception (coitus interruptus). Data were analyzed and P values ≤0.05 were considered statistically significant.Results: Rates of spontaneous and induced abortion, ectopic pregnancy, and vaginal bleeding during second half of pregnancy were more common in the removed IUD group compared to the withdrawal method, differences however not significant. No fetal abnormality was observed in IUD group. Preterm birth (p= 0.045), preterm premature rupture of membrane (p= 0.01), and vaginal bleeding during pregnancy (p= 0.01), were more prevalent in the IUD group (retained and removed) compared to those using the withdrawal method.Conclusions: Considering the adverse outcomes, we knew women with pregnancy after failure of IUD were at an increased risk for such outcomes, compared to those using the withdrawal method; however the results of this research showed these adverse effects are not significant when pregnancy with IUD is detected earlier and IUD is removed during the early stage(s) of pregnancy

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

Peer reviewedPublisher PD