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

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

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

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

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

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

BCG: the only available vaccine against tuberculosis: review article

Background: Despite advances in the vaccinology and chemotherapy in the past century, tuberculosis is still responsible for two million deaths every year. Emergence of multi-drug resistant strain and coinfection of TB-HIV make it a serious concern. Treatment and control of tuberculosis is a great health burden in every community. Active tuberculosis in children has very severe consequences especially those who are under 5-years-old, therefore vaccine indication should be taken. Bacille Calmette-Guérin (BCG) is a live attenuated strain of Mycobacterium bovis that has been used for providing immunity or protection against tuberculosis (TB). In addition, BCG provides relative protection against leprosy and Buruli ulcer, it also can be used for treatment of bladder cancer. BCG is the most widely administered vaccine around the world. It has been given to over three billion individuals over the past decades. At first it was developed in 1908 at the Pasteur Institute in Lille by Albert Calmette and Camille Guérin. In fact BCG is a strain of Mycobacterium bovis that bear deletion in its genome following too long subculture in special media. Deletion in region of deletion 1 (RD1), a specific region of Mycobacterium bovis genome, has decreased pathogenicity of BCG strain. Following culture of BCG on different media since 1921 make genetic variation in the BCG strains that have specific characteristics. BCG should begin given to only immune-competent individuals and should not be administered to immunocompromised people. This vaccine is not effective in people formerly infected or sensitized with environmental mycobacteria. Previous meta-analysis studies indicate that BCG has variable range of protection from 0 to 80 percent against pulmonary TB, but is very effective against severe disseminated forms such as meningitis and miliary form of TB. Despite many research and develop new generation vaccine against TB, BCG vaccine still remains as the only effective vaccine because many efforts to replace it with better ones were unsuccessful

No evidence of human papillomaviruses in non-genital seborrheic keratosis

Background: Seborrheic keratosis (SK) is a benign epidermal tumor of unknown etiology. Because of its wart-like morphology, Human papillomaviruses (HPVs) have been suggested as a possible causative agent. Viral involvement, however, has not been confirmed yet despite research and the association between HPVs and seborrheic keratosis has not been studied among Iranian population by PCR. Objectives: The aim of this case-control study was to evaluate the presence of HPVs DNA in non-genital SK by PCR. Materials and Methods: Fifty biopsy specimens obtained from patients with non-genital SK and 50 controls were analyzed using polymerase chain reaction (PCR). Results: No HPVs DNA was detected by PCR within the tissue extracts from paraffin-embedded SK samples, while one of the controls was HPVs DNA positive. The age range of the patients was 20 to 82 yrs (mean = 52). Twenty-eight patients (56%) were males and 22 patients (44%) were females. The most common anatomic site was the face. Histopathologic changes due to viral infection such as koilocytosis (10%), dyskeratosis (66%), mitosis (28%), and parakeratosis (88%) were evident within the lesions. The most common histologic type was acanthotic type. Conclusion: Our results showed that there is no association between HPVs and seborrheic keratosis in investigated subjects

Comparison of the prevalence of enteroviruses in blood samples of patients with and without unstable angina

BACKGROUND: Although the role of enteroviruses has been proved in heart diseases, extensive information is not available on the association between enteroviruses and unstable angina. In the present study, the authors compared the prevalence of enteroviruses in patients with and without unstable angina. METHODS: Blood samples were taken from 51 patients with unstable angina and 55 patients without unstable angina or myocardial infarction that were admitted to Imam Reza and Ghaem hospitals (Mashhad, northeast of Iran). Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers for the detection of the enteroviruses in blood samples of study subjects. RESULTS: Patients with and without unstable angina were similar in age with mean &plusmn; standard deviation of 62.6 &plusmn; 12.8 and 59.7 &plusmn; 12.7 years, respectively (P = 0.243) and there were no differences in gender in these two groups (P = 0.174). Prevalence of the enteroviruses in patients with unstable angina was higher only in 66-80 years age group compared to the control group (patients without unstable angina, P = 0.032). There was a higher prevalence of enterovirus RNA positivity in the blood samples of women with unstable angina (75.9%) than those without unstable angina (41.7%, P = 0.011), however, no significant difference was observed in men (P = 0.983). CONCLUSION: Our data showed that enteroviral RNA positivity was higher in patients with unstable angina compared to those without unstable angina. However, the differences between the two groups were not statistically significant. &nbsp;&nbsp;</p

The Frequency of VIM 2, 3, 9, 11 and VIM all among Metallo-beta-Lactamase Producing Pseudomonas aeruginosa

Introduction: Antibiotic resistance crisis has always been a serious problem for human health and many hospitalized patients are affected worldwide. Pseudomonas aeruginosa is a gram-negative pathogen and one of the most common causes of nosocomial infections. The main mechanism of resistance to beta-lactam antibiotics is the presence of metallo-beta-lactamase (MBL) enzymes. Most of the MBL genes are found in plasmids. The aim of this study was to evaluate the frequency of MBL-producing P. aeruginosa isolates caused by VIM-all and VIM 2, 3, 9, 11and16 genes.   Materials & Methods: Antimicrobial susceptibility of 127 clinical isolates of P. aeruginosa was determined using the standard Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standard Institute (CLSI). Combined-disk test was used for phenotypic determination of MBLs-producing isolates. After DNA extraction, VIM-all and in specific, VIM 2, 3, 9, 11 and 16 genes were amplified using PCR method.   Findings: A total Of 127 clinical isolates of P. aeruginosa, 62 isolates (49%) were resistant to imipenem and 31 isolates (24.5%) showed phenotypic evidences of MBL production. Moreover, among imipenem resistant strains VIM-all genes were found in 12.5% of cases, but the VIM 2-3-9-11 and 16 genes were not detected in samples.   Discussion & Conclusions: The results obtained in this study suggest that in P. aeruginosa, the highest antibiotic resistance observed was to cefazolin (98%) followed by nalidixic acid (91%) and the least resistance were to ciprofloxacin (31%). One of the reasons for this trend is the growth of antibiotic-resistant bacteria and the known mechanisms of bacterial resistance