In Silico approach: Design an Optimized shRNA against RUNX1 Gene to Target HIV.

INTRODUCTION: Human Immunodeficiency Virus (HIV) is a retrovirus with single-stranded RNA that leads to the challenging disease of acquired immunodeficiency syndrome (AIDS). Combination antiretroviral therapy (cART) can prevent the progression of the disease, but it is not capable of long-term HIV elimination. One of the significant obstacles to treating HIV-1-infected individuals is the creation of latent cell reservoirs early in the infection. Gene-based therapies that utilize RNA interference (RNAi) to silence host or viral gene expression are considered promising therapeutic approaches. It has been demonstrated that RUNX1, a T-cell-specific transcription factor, may significantly affect HIV replication and infection. According to accumulating evidence on the role of interfering RNA techniques in inhibiting gene expression and considering the role of RUNX1 in the replication of HIV-1. In this study, we aim to design shRNAs against RUNX1 that can target the replication of HIV-1.

Quantitative analysis of Epstein-Barr virus DNA in plasma and stomach biopsies of patients with gastric cancer.

Epstein-Barr virus (EBV) associated gastric carcinoma (EBVaGC) is a subtype of gastric cancer with distinct histological and molecular features. The study aimed to assess the EBV DNA copy number and the prevalence of EBVaGC in gastric cancer samples taken from Iranian patients. The next aim was to assess whether the DNA and microRNAs EBV are present in plasma. EBV load was analyzed in 68 gastric cancer biopsies and compared with the results of EBV-encoded small RNA in situ hybridization (EBER-ISH) test in these patients. After the detection of 6 EBV miRNAs in gastric tissue by stem-loop RT-PCR, plasma samples were evaluated for the viral load and EBV miRNAs. Four gastric cancer cases were EBER -ISH positive (5.8%), with a significantly higher viral load than the remaining cases, 47,781 vs. 1909 copies/µg of tissue DNA. Here, was also found a significant difference in plasma EBV load between EBER-positive and EBER-negative cases. Although EBV miRNAs were detectable in all the EBER-positive tumors, the test did not detect any of these miRNAs among the plasma samples tested. Our data indicate that the prevalence of EBVaGC among Iranian patients with gastric cancer is lower than the global prevalence and although none of the EBV miRNAs were detected in plasma, evaluation of EBV microRNAs in tumor tissue, especially miR-BART7-3p, may constitute useful biomarkers for diagnosis of EBVaGC.

Sequence variations of Epstein-Barr virus LMP1 gene in gastric cancer and chronic gastritis isolates from Iranian patients.

Aim: The current study aimed to investigate sequence variations in the C-terminus of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV) isolates from Iranian patients with chronic gastritis or gastric cancer (GC). Background: LMP1, an essential viral oncoprotein, is the critical element in the immortalization of B cells. It contains a small twenty-four amino acid cytoplasmic N-terminal region, six transmembrane segments, and a two hundred amino acid cytoplasmic C-terminal domain. Most LMP1-mediated signal transduction events are moderated by some functional parts of the cytoplasmic C-terminal domain. Methods: Thirty-two EBV-positive biopsy tissues were obtained from patients with gastric cancer and patients with chronic gastritis. The C-terminal nucleotide sequences of LMP1 were amplified using nested-PCR and analyzed by DNA sequencing. Results: Four to eight copies of the 11 repeat elements (codon 254-302) were observed in the carboxyl-terminal site of patients, but no relationship was found between the number of repeat sequences and disease status. The 30-bp deletion corresponding to codon 345-354 of the B95-8 strain was observed in 34% of isolates, and the remaining samples were non-deleted. In the gastric cancer group, a higher number of 33-bp repeats (≥5 repeats) was observed in 30-bp-deletion (100%) than in non-deleted (42%) isolates, and the difference was statistically significant. Analysis revealed that a gastritis isolate may be the result of recombination between Alaskan and China1 strains. Conclusion: Overall, the current results showed no association between C-terminal sequence variations of LMP1 and malignant or non-malignant isolate origin.

Detection and quantification of Epstein-Barr virus, cytomegalovirus, and human herpesvirus-6 in stomach frozen tissue of chronic gastritis and gastric cancer patients.

Human herpes viruses (HHVs) are among the most common infectious agents detected in the gastrointestinal tract that might be involved in oncogenesis and other gastrointestinal disorders. Although the link between the Epstein-Barr virus (EBV) and gastric cancer (GC) has been established, the role of the viruses in various stomach diseases remains unknown. The frequencies and viral copy number of EBV, cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) among 50 gastric cancer tumors and 105 chronic gastritis tissues were measured by quantitative real-time PCR. In the tumor specimens and the adjacent normal tissues EBV was found in 60% and 30.9%, CMV in 14% and 4.7%, and HHV-6 in 18%, and 14.2%, respectively. The detection rate of EBV and CMV was found to be significantly higher in tumor tissues relative to the adjacent normal tissues. Also, in chronic gastritis, the frequency of EBV, CMV, and HHV-6 was 19%, 12.3%, and 15.2%, respectively, compared with 16.4%, 1.1%, and 8.2% in their corresponding normal tissues. Here, the CMV frequency was found to be significantly higher in gastritis tissues relative to the adjacent normal tissues. Furthermore, viral load in both gastric cancer and gastritis groups was higher in either tumor or gastritis lesion compared with matched adjacent normal tissue. This study showed a clear association between gastric cancer with both EBV and CMV. Meanwhile, analyses revealed a strong association between the EBV, CMV, and HHV-6 viral loads with gastritis (P = 0.0026, P < 0.0001, and P = 0.0405, respectively). Our results suggest that these three viruses might contribute to the induction and development the gastritis and gastric cancer.

Evaluation of IL-1ß and IL-6 Expression following EBNA-1 and BRLF-1 Peptide Treatment in Epstein-Barr Virus-Positive Multiple Sclerosis Patients.

INTRODUCTION: Epstein-Barr virus (EBV/HHV-4) has been implicated in the pathogenesis of multiple sclerosis (MS). This study was conducted to investigate the levels of pro-inflammatory cytokines IL-1ß and IL-6 in healthy EBV carriers and MS patients with prior EBV infection in response to treatment with EBV nuclear antigen 1 (EBNA-1) and replication and transcription activator (BRLF-1/Rta) peptide antigens in whole blood cell culture to assess the cytokine expression across all cells in the peripheral blood. METHODS: Isolated whole blood cells from the included participants were incubated at a concentration of 106 cells/mL with BRLF-1 or EBNA-1. The amount of IL-1ß and IL-6 transcripts were measured with quantitative RT-PCR at day 3 after incubation. MTT assay was conducted to examine cytotoxicity of the peptides and their effect on cell viability. Changes in cytokine expression and cell viability were analyzed using one-way and two-way ANOVA, respectively. RESULTS: Ten MS patients and ten healthy donors were enrolled in the study. Treatment with the peptide antigens resulted in increased cytokines expression in both MS patients and healthy subjects. Furthermore, IL-1ß levels were higher in MS patients compared to healthy EBV carriers. MTT assay revealed no significant difference in cell viability between the two groups. DISCUSSION: The higher levels of IL-1ß in response to EBV antigens in MS patients may reflect the host neuroinflammatory environment and support the notion that immune response against EBV has a role as an aggravating factor in the progression of MS by contributing to the neuroinflammatory cascade.

Serotype-dependent recombinant adeno-associated vector (AAV) infection of Epstein-Barr virus-positive B-cells, towards recombinant AAV-based therapy of focal EBV + lymphoproliferative disorders.

BACKGROUND: B-cell proliferative disorders, such as post-transplant lymphoproliferative disease (PTLD), are increased among persons afflicted by T-cell compromise. Most are Epstein-Barr virus (EBV) + and can first present with a focal lesion. Direct introduction of oncolytic viruses into localized tumors provides theoretical advantages over chemotherapy, immunotherapy and radiation therapy by reducing systemic toxicity. Despite extensive study as a vehicle for gene therapy, adeno-associated viruses (AAV) have rarely been applied to human cancer research due to technical and theoretical obstacles. Moreover, human B-cells have historically been described as resistant to AAV infection. Nonetheless, advances using different recombinant (r)AAV serotypes with unique tropisms to deliver cytotoxic therapy suggested a localized anti-tumor approach was feasible. METHODS: As a prelude to the development of a therapeutic vehicle, the ability of fifteen distinct EGFP-bearing rAAV serotypes to transduce human B-cells, including primary, immortalized, and B-cell tumor lines ± EBV was assessed by confocal microscopy, flow cytometry and subsequently cell viability assay. RESULTS: Rank order analysis revealed augmented transduction by rAAV6.2 and closely related virions. EBV infection of EBV-negative B-cell tumor lines and EBV immortalization of primary B-cells increased susceptibility to rAAV6.2 transduction. As a proof of concept, transduction by rAAV6.2 encoding herpes simplex virus type 1 (HSV1)-thymidine kinase (TK) eliminated TK-negative rhabdomyosarcoma cells and diminished viability of transduced B-cell lines upon incubation with ganciclovir. CONCLUSIONS: rAAV serotypes differentially transduce human B-cell lines reversing the dogma that human B-cells are refractory to AAV infection. EBV + B-cells display increased susceptibility to rAAV6.2 infection, uncovering a new method for improved nucleic acid transfer into transfection-resistant B-cell lines. The introduction of a functional suicide gene into the rAAV6.2 genome identifies a candidate vector for the development of rAAV-based oncolytic therapy targeting focal EBV-bearing B-lymphoproliferative disorders.

Investigation of IL-2 and IFN-γ to EBV Peptides in Stimulated Whole Blood among Multiple Sclerosis Patients and Healthy Individuals.

INTRODUCTION: Epstein-Barr virus (EBV), a double-stranded DNA virus, has 2 phases of lytic and latent infection in host cells. After infecting B lymphocytes, EBV becomes persistent in these cells. In healthy individuals, T lymphocytes play a key role in killing EBV-infected B cells. Statistical studies have shown that symptomatic EBV infection increases the risk of MS. METHODS: This study intended to measure the immune system's response against the different components of EBV, focusing particularly on T lymphocytes' reaction. Consequently, the mRNA level of IL-2 and IFN-γ, liable for impressing autoimmune diseases and as indicators of T-cell function, was compared in EBNA1- and BRLF1-treated whole blood (WB) cultures of 10 healthy individuals and 10 MS patients using real-time RT-PCR. RESULTS: The analysis of the results demonstrated a significant increased level of IL-2 in MS patients than healthy subjects after exposure to both peptides. Also, the mRNA level of IFN-γ increased in MS patients in EBNA1-treated WB culture. CONCLUSION: According to the study's results, EBV peptides can reactivate immune cells, especially T lymphocytes, and may indirectly induce inflammation and develop MS; however, it seems that long-time exposure to these peptides has reducing effect on T-cell function and faces the control of infected B lymphocytes with difficulties.

Brain HIV-1 latently-infected reservoirs targeted by the suicide gene strategy.

Reducing the pool of HIV-1 reservoirs in patients is a must to achieve functional cure. The most prominent HIV-1 cell reservoirs are resting CD4 + T cells and brain derived microglial cells. Infected microglial cells are believed to be the source of peripheral tissues reseedings and the emergence of drug resistance. Clearing infected cells from the brain is therefore crucial. However, many characteristics of microglial cells and the central nervous system make extremely difficult their eradication from brain reservoirs. Current methods, such as the "shock and kill", the "block and lock" and gene editing strategies cannot override these difficulties. Therefore, new strategies have to be designed when considering the elimination of brain reservoirs. We set up an original gene suicide strategy using latently infected microglial cells as model cells. In this paper we provide proof of concept of this strategy.

Immune Response of Gamma-Irradiated Inactivated Bivalent Polio Vaccine Prepared plus Trehalose as a Protein Stabilizer in a Mouse Model.

INTRODUCTION: Poliovirus causes paralysis by infecting the nervous system. Currently, 2 types of polio vaccine are given in many countries in polio eradication program including inactivated polio vaccine (IPV) and oral polio vaccine (OPV). Because of OPV-related paralysis, OPV should be replaced by IPV. METHODS: The aim of this study was to prepare the gamma-irradiated IPV and determine its effectiveness compared with the commercial vaccine (OPV) in the mouse model. The virus titration of OPV was determined and then inactivated by the appropriate dose of gamma radiation into an irradiated vaccine formula. The vaccine was inoculated in BALB/c mice in 2 different formulations of intramuscular injection with 2-week intervals. The level of anti-polio-neutralizing antibody and polio-specific splenocyte proliferation assay were evaluated by collecting the blood samples and spleens of the vaccinated groups with conventional vaccine and irradiated vaccine. RESULTS: There was a significant increase in the neutralizing antibody titration between all of the vaccinated groups and negative control group (A) (p < 0.05). And it shows that the IPV by gamma irradiation has the highest antibody titration. Also, the increasing of stimulation index value in the B* group, F group, and G group was the most against other groups. Furthermore, the neutralizing anti-serum titer and splenic lymphocyte proliferation assay show humoral and cellular immunity were significantly increased in the irradiated vaccine group as compared with conventional group. CONCLUSION: According to the results, gamma-irradiated IPV could induce humoral and cellular immunity in vaccinated mouse groups, so the irradiated poliovirus could be recommended as a good candidate vaccine to prevent the transport of poliovirus to the central nervous system and thus protect against paralysis.

High prevalence of occult hepatitis B infection (OBI) among healthy children and their parents in Alborz province, Iran; Vertical OBI, myth or truth?

BACKGROUND: Occult hepatitis B infection (OBI) has been described in various clinical settings including after hepatitis B virus (HBV) immunization. The purpose of study was to characterize the prevalence of OBI among immunized children from a subset of general population and the parents of OBI-positive cases. METHODS: Sera of 1200 children from general population who have been previously immunized by HBV vaccine were assayed for anti-HBs. 660 were randomly selected for HBV DNA testing by different polymerase chain reaction (PCR) methods and were analysed by direct sequencing on surface genes. RESULTS: None of participants were positive for HBsAg and anti-HBc. 549 (45.7%) and 651 (54.3%) cases had anti-HBs > 10 mIU/mL (responders) and < 10 mIU/mL (nonresponders) respectively. Of 660 selected specimens, 91 (16%) of children were positive for OBI. 23 (25.2%) and 68 (74.8%) of HBV DNA positive cases were belonged to responders and nonresponders, respectively, showing significant difference (P < .001). The mean levels of anti-HBs in OBI-positive and OBI-negative groups, showed no considerable variations. The mean viral load for OBI-positive cases showed substantial differences between responders and nonresponders (P = .007). Of 49 parents (98 individuals) of OBI-positive children 11 (22%) and 18 (36%) were positive for anti-HBc and anti-HBs respectively. Molecular testing was positive in 32 subjects (16 couples, 32.6%). In total, 6 mothers and 11 fathers were positive for OBI. CONCLUSION: A proportion of OBI-positive vaccinated children could be existed in different populations. This finding could be arisen from vertical HBV transmission or vertical OBI possibly from their parents.

Signature of natural resistance in NS3 protease revealed by deep sequencing of HCV strains circulating in Iran.

A tremendous upscale of screening and treatment strategies is required to achieve elimination of the hepatitis C virus (HCV) in Iran by 2030. Among treated patients, at least 5-10% is expected to experience treatment failure. To efficiently retreat cases with prior exposure to NS5A and NS5B drugs, knowledge on the natural prevalence of NS3 resistance is key. The NS3 region of 32 samples from sixteen Iranian HCV patients, among which 6 injecting drug users, was amplified and subjected to deep sequencing. Amplification and sequencing were successful in 29 samples. The reads were assembled to consensus sequences and showed that 6 patients were infected with HCV1a (37.5%), 7 with HCV1b (43.8%) and 3 with HCV3a (18.7%). Nucleotide identities were shared for >97% between intra-host sequences. Two patients were infected with natural resistant viruses, of which one solely comprising low frequency variants. Inferred phylogenies showed that Iranian sequences clustered together for HCV1a and HCV1b, while for HCV3a a potential recombination event was detected. We firstly report the use of deep sequencing for HCV in Iran, demonstrate the use of NS3 inhibitors as salvage therapy in case of retreatment and stress the importance for Iran to prioritize drug users for screening and treatment.

Next-generation sequencing for the clinical management of hepatitis C virus infections: does one test fits all purposes?

While the prospect of viral cure is higher than ever for individuals infected with the hepatitis C virus (HCV) due to ground-breaking progress in antiviral treatment, success rates are still negatively influenced by HCV's high genetic variability. This genetic diversity is represented in the circulation of various genotypes and subtypes, mixed infections, recombinant forms and the presence of numerous drug resistant variants among infected individuals. Common misclassifications by commercial genotyping assays in combination with the limitations of currently used targeted population sequencing approaches have encouraged researchers to exploit alternative methods for the clinical management of HCV infections. Next-generation sequencing (NGS), a revolutionary and powerful tool with a variety of applications in clinical virology, can characterize viral diversity and depict viral dynamics in an ultra-wide and ultra-deep manner. The level of detail it provides makes it the method of choice for the diagnosis and clinical assessment of HCV infections. The sequence library provided by NGS is of a higher magnitude and sensitivity than data generated by conventional methods. Therefore, these technologies are helpful to guide clinical practice and at the same time highly valuable for epidemiological studies. The decreasing costs of NGS to determine genotypes, mixed infections, recombinant strains and drug resistant variants will soon make it feasible to employ NGS in clinical laboratories, to assist in the daily care of patients with HCV.

Promoter DNA Methylation and mRNA Expression Level of p16 Gene in Oral Squamous Cell Carcinoma: Correlation with Clinicopathological Characteristics.

The aim of this study was to investigate the relationship between p16 methylation and its expression in oral squamous cell carcinoma (OSCC). Also the contribution of clinicopathological factors, HPV infection and smoking in p16 expression and promoter methylation has been investigated. In this study 67 consecutive OSCC patients and 59 normal individuals were enrolled. All patients were candidates for surgery of oral cavity and fresh tumor biopsies were collected and processed for DNA and RNA extraction. Normal gingival tissues were collected from individuals referred to dentistry clinic and considered as controls. All the cases and controls were checked for HPV infection and then promoter methylation and expression of p16 gene were determined using Methylation-specific PCR (MSP) and real-time PCR (QPCR), respectively. Methylation of p16 in tumors and normal tissues were 59.7 and 38.9%, respectively. Most of hypermethylated samples (>82%) were in high grades. P16 methylation was comparable in HPV+ and HPV- patients or smokers. P16 was overexpressed (~3 fold; p = 0.044) in HPV+ tumors, but it was significantly down-regulated in smoker patients (40% of all tumors). Comparison of P16 expression in OSCC tumors with different degrees of promoter methylation further suggest the relationship of methylation rate and down-regulation of P16 expression. The p16 methylation and expression was differentially affected in patients with HPV infection and the smoker cases. Regardless of the influence of environmental factors, it appears that P16 status is useful for classifying patients with OSCC and for influencing treatment strategies in accordance with this classification. Moreover, targeting the upregulation of p16 could be a promising therapeutic option.

Design and Development of a Quantitative TaqMan Real-Time PCR Assay for Evaluation of HIV-1 (group M) Viral Load in Plasma Using Armored RNA Standard.

BACKGROUND: Human immunodeficiency virus-1 (HIV-1) is a viral infectious agent that gradually extinguishes the immune system, resulting in the acquired immune deficiency syndrome (AIDS). The aim of this study was to develop a TaqMan based detection assay to evaluate HIV-1 plasma viral load and to construct a stable internal positive control (IPC) and external positive control (EPC) RNA based on Armored RNA (AR) technology. METHODS: The MS2 maturase, coat protein gene and HIV-1 pol gene were cloned in pET-32a plasmid. The recently fabricated recombinant plasmid was transformed into Escherichia coli strain BL2 (DE3) and protein expression and Armored RNA was fabricated in presence of isopropyl-L-thio-D-galactopyranoside (IPTG). The Armored RNA was precipitated and purified by polyethylene glycol (PEG) and sephacryl S-200 chromatography. The stability of Armored RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy (TEM) and gel agarose electrophoresis. The specificity, sensitivity, inter- and intra-day precision, and the dynamic range of the assay were experimentally determined. RESULTS: The AR was stable in presence of ribonuclease, and the assay had a dynamic detection range from 101 to 105 copies of AR. The coefficient of variation (CV) was 4.8% for intra-assay and 5.8% for inter-assay precision. Clinical specificity and sensitivity of the assay were assessed at 100% and 96.66%, respectively. The linear regression analysis confirmed a high correlation between the in-house and the commercial assay, Real Star HIV-1-qRTPCR, respectively (R2 = 0.888). CONCLUSIONS: The AR standard is non-infectious and highly resistant in the presence of ribonuclease. The TaqMan assay developed is able to quantify HIV viral load based on a novel conserved region of HIV-1 pol gene which has minimal sequence inconsistency.

Preparation and Evaluation of Ribonuclease-Resistant Viral HIV RNA Standards Based on Armored RNA Technology

BAckground: The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model. Methods: The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography. Results: The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33. Conclusion: Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory.

Induction of Immune Response and Protective Immunity by a Local Isolated Varicella Virus in Animal Model: A Future Candidates for Vaccine Production.

Preparation of the indigenous varicella zoster vaccine could significantly reduce the disease burden of varicella zoster virus especially in immunosuppressed children. To achieve this goal, the varicella zoster virus was isolated from an 8 years boy infected with chicken pox. The virus was cultivated in sensitive cell line and determined varicella zoster. The adaptation and attenuation of virus was carried out after several passages in MRC-5 cell culture, Primary Guinea pig embryo fibroblast cell culture and again switching in MRC-5 cell culture. The challenged of vaccine dose was found 3LogCCID50. Following two doses of immunization in guinea pigs via inoculated cell culture-fluid attenuated- local isolated VZV at zero and 14 day, the humoral immune response, varicella-zoster virus (VZV) IgG and IgM were determined using enzyme-linked Immunosorbent and seroneutralization assays at 7, 14, 21, 30, 60, 90.120 days after receiving of the first and second dose of vaccine. The results of immunization showed good 93% seroconversion in guinea pig which compared with vOKa vaccine was not significant (p<0.05). The prepared attenuate varicella zoster virus promising a candidate Virus for our future plan to vaccine production.

Effect of TGF-ß/smad signaling pathway blocking on expression profiles of miR-335, miR-150, miR-194, miR-27a, and miR-199a of hepatic stellate cells (HSCs).

AIM: The aim of this study was to determine the effect of inhibition of TGF-ß/smad signaling on the expression profiles of miR-335, miR-150, miR-194, miR-27a, miR-199a of hepatic stellate cells (HSCs). BACKGROUND: Liver fibrosis is excessive deposition of extracellular matrix proteins due to ongoing inflammation and HSC activation that occurs in most types of chronic liver diseases. Recent studies have shown the importance of microRNAs in the pathogenesis of chronic liver diseases. METHODS: In this study, for inhibition of TGF-ß smad-signaling pathway, expressing Smad4 shRNA plasmids were transfected into HSCs. Subsequently, using Real Time-PCR, we measured the expression levels of miR-335, miR-150, miR-194, miR-27a and miR-199a. RESULTS: Gene expression analysis showed that downregulation of Smad4 by vector Smad4shRNA significantly increased the expression levels of miR-335 (P<0.01) and miR-150 (P<0.001) and decreased the expression level of miR-27a (P<0.05). CONCLUSION: The results of this study suggest that blocking TGF-ß smad-signaling can also differentially modulate microRNA expression in support of activation and fibrogenesis of HSCs.

E6-Specific Detection and Typing of Human Papillomaviruses in Oral Cavity Specimens from Iranian Patients

Background: Detection and quantification of human Papillomavirus (HPV) genome in oral carcinoma play an important role in diagnosis, as well as implications for progression of disease. Methods: We evaluated tissues from 50 esopharyngeal cancers collected from different regions of Iran for HPV E6 using the two type-specific primers sets. E6 gene of HPV genotypes was amplified by specific primers. The sensitivity of PCR assay was analyzed and determined using HPV-DNA-containing plasmids. Real-time PCR was utilized to determine the prevalence and HPV viral load in patients with oral cavity squamous cell carcinoma. Results: Eighteen (36%) specimens were positive for HPV. Among the 18 positive specimens, 10 showed HPV-18 (55.55%), and 8 specimens were positive for HPV-11 (44.44%). Of the 18 infected specimens, 6 (33.32%) and 12 (66.65%) were identified as high-titer and low-titer viral load, respectively. Conclusion: The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies.

The effect of the hepatitis C virus (HCV) NS3 protein on the expression of miR-150, miR-199a, miR-335, miR-194 and miR-27a.

Hepatitis C virus (HCV) infection is considered one of the most important causes of chronic liver diseases. Many reports have shown that the proteins of the HCV via interactions with gene expression regulatory networks such as cellular pathways and microRNAs can contribute to the development of chronic liver diseases. The present study aimed to investigate the effects of the HCV NS3 protein on the expression of miR-150 miR-199a, miR-335, miR-194, miR-27a in a cell culture model. Plasmids expressing the full length of the HCV NS3 protein were transfected into the LX-2 cell line, while at the same time a plasmid expressing empty GFP (green fluorescent protein) was used as a negative control group. Subsequently, total RNA was extracted and real-time PCR was performed to measure microRNA expression levels. Additionally, the trypan blue exclusion test was performed to examine the effect of the expressing NS3 protein plasmid on cellular viability. The analysis of microRNA gene expression in LX-2 cells indicated that the NS3 protein, which is endogenous to HCV, can significantly upregulate the expression of miR-27a and downregulate the expression of miR-335 and miR-150 in comparison with the control plasmid expressing GFP and normal cells (p < 0.01). These results suggest that the HCV NS3 protein may play a role in the pathogenesis of chronic hepatic diseases such as liver fibrosis via interaction with cellular microRNAs and modulation of microRNA gene expressions.