Molecular genetic study of glutaric aciduria, type I: Identification of a novel mutation.

Glutaric acidemia type I (GA-1) is an inborn error of metabolism due to deficiency of glutaryl-CoA dehydrogenase (GCDH), which catalyzes the conversion of glutaryl-CoA to crotonyl-CoA. GA-1 occurs in about 1 in 100 000 infants worldwide. The GCDH gene is on human chromosome 19p13.2, spans about 7 kb and comprises 11 exons and 10 introns. Tandem mass spectrometry (MS/MS) was used for clinical diagnosis in a proband from Iran with GA-1. Sanger sequencing was performed using primers specific for coding exons and exon-intron flanking regions of the GCDH gene in the proband. Cosegregation analysis and in silico assessment were performed to confirm the pathogenicity of the candidate variant. A novel homozygous missense variant c.1147C > A (p.Arg383Ser) in exon 11 of GCDH was identified. Examination of variant through in silico software tools determines its deleterious effect on protein in terms of function and stability. The variant cosegregates with the disease in family. In this study, the clinical and molecular aspects of GA-1 were investigated, which showed one novel mutation in the GCDH gene in an Iranian patient. The variant is categorized as pathogenic according to the the guideline of the American College of Medical Genetics and Genomics (ACMG) for variant interpretation. This mutation c.1147C > A (p.Arg383Ser) may also be prevalent among Iranian populations.

Evaluation of Interleukin-21, 23 and 27 mRNA Expression and Protein Level in Liver Transplant Patients.

Cytokines have prominent roles in activating of different T cells and shifting the immune response, in this study the role of three cytokines (IL-21, IL-23 and IL-27) is investigated in the liver transplant rejection. Three EDTA-treated blood samples were collected from each liver transplanted patient in 1st, 4th and 7th day of post-transplantation. The expression level of the mentioned cytokines was determined using real-time PCR for all samples. Also, the serum levels of cytokines were determined using ELISA tests. In acute rejection (AR) group (51 patients), mRNA expression pattern of IL-21and IL-23 showed a steady increase, but this pattern was converse for IL-27. Our results in non-acute rejection (non-AR) group (54 patients) showed an elevation in day 4 and then a decrease in day 7 for IL-21 and IL-23 genes. This pattern was converse again for IL-27 gene. In comparison between the two groups, in all 3 sampling times the mean of mRNA expression level of IL-21 and IL-23, showed an increase in AR group which this increase was significant for IL-21 in the 3rd (p=0.007) and for IL-23 in 2nd (p=0.048) and 3rd (p=0.049) sampling time, but the pattern of mRNA expression for IL-27 was contrary to the results of IL-21 and IL-23. Furthermore, ELISA technique also, showed the serum level changes the same as cytokines. In this study IL-21 and IL-23 showed pro-inflammatory properties in the liver transplant rejected patients. Also, IL-27 having different expression pattern, showed anti-inflammatory behavior which needs more considerations in future.

Simulated Microgravity Condition Alters the Gene Expression of some ECM and Adhesion Molecules in Adipose Derived Stem Cells.

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.

Determining the Risk of Intra-Community Transmission of Tuberculosis in the Northwest of Iran Through 15 Loci Miru-Vntr Typing.

This study was carried out in order to investigating the effect of travelling on the transmission of tuberculosis from high- to low-burden TB countries. Mycobacteria samples isolated from patients of distinct and relatively co-related countries (Azerbaijan Republic and Tabriz [located in the northwest of Iran]) were analyzed through 15 loci MIRU-VNTR typing method. PCR was done using special primers for each of the loci; then the number of allele repeats for all loci were determined by the size of their fragments. Finally, the created numeric patterns for each isolate were analyzed and clustered, using MIRU-VNTRplus.org website. All 119 isolates dispersing at 106 distinct patterns were composed of 10 clusters with 23 members and 96 unique patterns. Nine and five loci had high and moderate discriminatory power, respectively, but only one of them was poor in clustering. The study showed that 89.08% of TB cases involved resulted from the reactivation pattern and 10.92% were related to ongoing transmission. Although Azerbaijan Republic is a higher-burden TB region than Tabriz and Azerbaijan people make frequent tours to Tabriz to receive low or free medical services, the findings showed no TB transmission from the regions at least during the year of the study.

Effect of Simulated Microgravity Conditions on Differentiation of Adipose Derived Stem Cells towards Fibroblasts Using Connective Tissue Growth Factor.

Background: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiating into a variety of mesenchymal tissues including osteoblasts, adipocytes and several other tissues. Objectives: Differentiation of MSCs into fibroblast cells in vitro is an attractive strategy to achieve fibroblast cell and use them for purposes such as regeneration medicine. The goal of this study was investigate the simulated microgravity effect on differentiation of Adipose Derived Stem Cells (ADSCs) to fibroblasts. Materials and Methods: To fibroblast differentiation 100 ng.mL-1 of connective tissue growth factor (CTGF), and for simulation microgravity, 2D clinostat was used. After isolation the human ADSCs from adipose, cells were passaged, and at passages 3 they were used for characterization and subsequent steps. After 7 days of CTGF and simulated microgravity treatment, proliferation, and differentiation were analyzed collectively by MTT assay, quantitative PCR analyses, and Immunocytochemistry staining. Results: MTT assay revealed that CTGF stimulate the proliferation but simulated microgravity didn't have statistically significant effect on cell proliferation. In RNA level the expression of these genes are investigated: collagen type I (COLI), elastin (ELA), collagen type III (ColIII), Matrix Metalloproteinases I(MMP1), Fibronectin 1 (FN1), CD44, Fibroblast Specific protein (FSP-1), Integrin Subunit Beta 1 (ITGB1), Vimentin (VIM) and Fibrillin (FBN). We found that expression of ELN, FN1, FSP1, COL1A1, ITGB1, MMP1 and COL3A1 in both condition, and VIM and FBN1 just in differentiation medium in normal gravity increased. In protein level the expression of COL III and ELN in simulated microgravity increased. Conclusions: These findings collectively demonstrate that the simulated microgravity condition alters the marker fibroblast gene expression in fibroblast differentiation process.

Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells.

CONTEXT: Lung cancer is one of the most serious types of cancer that often diagnosed at advanced stage. MicroRNAs (miRNAs) are small non-coding molecules which silence gene expression of target gene (s) at posttranscriptional level. They are key regulators of cell cycle, apoptosis, anti-cancer drug responsiveness and metastasis. AIMS: Identification of the differential expression level of miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 in A549 cell line versus normal tissues and their correlation with selected corresponding target genes. MATERIALS AND METHODS: A549 cell line was cultured in F-12K medium and miRNA was extracted from normal tissues (2-3 cm adjacent to tumor tissue) and A549 cell line. cDNA was synthesized with specific stem-loop primers for each miRNA, while OligodT primer was used for target genes cDNA synthesis. Real-time quantitative polymerase chain reaction. (RT-qPCR) was used to analyze the expression pattern of miRNAs and target genes in A549 and normal non-small cell lung carcinoma. (NSCLC) tissues. RESULTS: miR-15a/16, miR-34a, miR-126 and miR-128 were down-regulated significantly. (>2-fold change), while miR-21 and miR.210 were up-regulated in A549. Bcl-2 as miR-34a target gene was down-regulated while Hif-1α and Akt-3 were up-regulated that might be miR-210 and miR-34a target genes, respectively. CONCLUSION: The significant differential expression level of these miRNAs made them as candidate biomarkers in NSCLC tumor tissues of patients. Perhaps Bcl-2 down-regulation and Akt-3 up-regulation can be linked with survival signals in A549 cell line. We can conclude that Bcl-2 and Akt-3 might be therapeutic targets to inhibit cell proliferation in NSCLC.

IL-17 mRNA expression and cytomegalovirus infection in liver transplant patients.

OBJECTIVES: Cytomegalovirus (CMV) establishes a lifelong, asymptomatic infection in immunocompetent hosts. Interleukin-17 producing CD4+ T-cells (Th-17) are a subtype of CD4+ T-cells. The precise role of Th-17 responses during cytomegalovirus replication has not been elucidated, although recent studies suggest that infections such as murine cytomegalovirus induce a Th-17 response. Th-17 cells also have been associated with allograft rejection and autoimmune diseases. In this study, we tried to find the relation of cytomegalovirus infection and interleukin 17 (IL-17) cytokine in liver-transplanted patients. MATERIALS AND METHODS: Two groups of patients were evaluated in this study. The first group consisted of 54 cytomegalovirus uninfected livertransplanted patients, and the second group consisted of 15 cytomegalovirus-infected patients. Three ethylenediaminetetraacetic acid-treated blood samples were collected from each patient on days 1, 4 and 7 post liver transplant. For diagnosing cytomegalovirus infection antigenemia and Taq-Man real-time polymerase chain reaction protocols were used. Also, to determine the expression level of IL-17 gene, an in-house SYBR green real-time polymerase chain reaction technique was used. RESULTS: Using antigenemia and also Taq-Man real-time polymerase chain reaction helps find active cytomegalovirus infection, and the load of cytomegalovirus in each patient. The first group of patients showed that IL-17 expression level was down-regulated after day 4 of sampling. But in cytomegalovirus-infected patients, IL-17 expression level was increased significantly. The results between IL-17 gene expression level between the 2 groups of patients showed that IL-17 expression level significantly increased in second group during day 4 (P = .038) and 7 (P = .009) postliver transplant. CONCLUSIONS: Significant increase of IL-17 mRNA levels in cytomegalovirus-infected group compared with the uninfected one reinforced the role of IL-17 as a proinflammatory cytokine dealing with cytomegalovirus infection in liver transplanted patients.

Expression of miRNAs in non-small-cell lung carcinomas and their association with clinicopathological features.

Lung cancer is recognized as a leading cause of cancer-related deaths worldwide. Over the past several years, evidence emerged that microRNAs (miRNAs), a class of small non-coding RNA molecules regulating gene expression at posttranscriptional level, play an important role in cell functioning, as well as in human diseases. Here, we analyzed expression of miR-15a/16, miR-21, miR-34a, miR-126, miR-128, and miR-210 at transcriptional level in 30 non-small-cell lung carcinoma (NSCLC) tumor tissues compared to the matched adjacent normal tissues and their correlation with clinicopathological features of the patients. Samples were collected from the NSCLC patients undergoing surgery before radiotherapeutic or chemotherapeutic treatment. Expression levels of miRNAs were assessed by TaqMan RT-PCR assay. The data obtained in this study were processed using REST 2009 and SPSS statistical software. The graphs were designed by GraphPad prism 5.0. In tumor samples, we found downregulation of miR-15a/16 (50/83.3%), miR-34a (83.3%), miR-126 (70%), and miR-128 (63.3%). At the same time, miR-21 and miR-210 were upregulated by 53.3 and 66.6% in cancer tissue versus matched adjacent normal tissues, respectively. No significant correlation was found between the expression levels of miR-15a/16, miR-21, miR-34a, miR-126, miR-128, and miR-210 and lymph node, tumor size, sex, and smoking. However, the study demonstrated a correlation between a change in expression of miR-15, miR-16, miR-34a, miR-126, and miR-210 compared to normal tissues and TNM staging (P < 0.05). Furthermore, miR-126 expression level was different in adenocarcinomas and squamous cell carcinoma (SCC) subtype (P < 0.1). Detailed analysis revealed significant change in expression of miR-15a/16, miR-34a, miR-126, and miR-210 in NSCLC tumor samples indicating involvement of these miRNAs in lung cancer pathogenesis. miR-210 demonstrated the most consistent increase in tumor tissues between different patients, suggesting its potential significance for NSCLC.

Cloning and expression of the functional human anti-vascular endothelial growth factor (VEGF) using the pcDNA3.1 vector and the human chronic myelogenous leukemia cell line K562.

In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.

Investigating GSTT1 and GSTM1 null genotype as the risk factor of diabetes type 2 retinopathy.

BACKGROUND: Diabetes is one of the multifactorial disorders with genetics and environmental factors playing important role in its cause. In diabetes, the defects in cellular metabolism results in increasing free radicals. These radicals react with other vital cellular molecules which are responsible in diabetes side effects. Human glutathione S-transferases (GST) are a family of enzymes that catalyses conjugation of electrophilic substances with glutathione. In this research the deletion of two of the most important genes of this family; GSTT1 and GSTM1 genes was investigated as the risk factor for diabetes mellitus type II and one of its most important complications; retinopathy. MATERIAL AND METHODS: In this study deletion of GSTT1 and GSTM1 genes in 57 diabetics' patients with retinopathy and 58 diabetic peoples without retinopathy was examined. DNA was extracted from peripheral blood and then multiplex PCR was performed following agarose gel electrophoresis to detect GSTT1 and GSTM1 null genotypes. Data were analyzed with SPSS v16 software. RESULTS: The results indicated that there was significant relationship between GSTM1 null genotype with retinopathy side effect of diabetes type 2. While there was no significant relationship between GSTT1 null genotypes with retinopathy in diabetes type 2. CONCLUSION: Significant correlation between GSTM1 null genotype and retinopathy in this and other studies could indicate this fact that impair cellular metabolism result in increase free radicals and oxidative pressure. Therefore, GST null genotypes may result in decrease antioxidant capacity which causes side effects of diabetes. Considering the performance of different classes of GST null genotypes additional studies are required to confirm this study.

Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria.

BACKGROUND: Toxoplasmosis is a worldwide-distributed infection which is mostly asymptomatic but can cause serious health problems in congenitally-infected newborns and immunecompromised individuals. Research is undergoing both to improve Toxoplasma serological tests, which play the main role in laboratory diagnosis of the infection, and develop an effective vaccine to prevent the infection. Some studies showed usefulness of rhoptry protein 1 (ROP1) antigen of Toxoplasma gondii (T. gondii) in serodiagnosis of the infection and induction of protective immunity. The purpose of this study was to produce recombinant ROP1 and evaluate its antigenicity against human infected sera. METHODS: DNA encoding ROP1, amino acids 171 to 574, was obtained from T. gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-15b. rROP1 was expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. RESULTS: DNA sequencing showed 99% similarity between the cloned sequence and the corresponding sequence in Gene bank. Results indicated the proper antigenicity of rROP1. Sera from Toxoplasma infected individuals specifically recognized rROP1 in Western blotting. CONCLUSION: rROP1 is antigenic toward human infected sera and can be used in studies for development of both a Toxoplasma serological test and a protective vaccine.

The prevalence of common CFTR mutations in Iranian infertile men with non-CAVD obstructive azoospermia by using ARMS PCR techniques.

PURPOSE: To evaluate five common cystic fibrosis trans-membrane conductance regulator (CFTR) mutations (ΔF508, G542X, R117H, W1282X and N1303K) in the Iranian infertile men with noncongenital absence of vas deferens (CAVD) obstructive azoospermia. METHODS: The common CFTR gene mutations were tested on blood samples from 53 infertile men with non-CAVD obstructive azoospermia and 50 normal men as control individuals. Genomic DNA is extracted from the whole blood and the common CFTR mutations have been detected by the amplification refractory mutation system (ARMS) techniques. RESULTS: The common CFTR mutations were found positive in 5/53)9.43%(for ΔF508 and 4/53)7.55%(for G542X mutation of all patients tested. Also, no CFTR mutations were detected in the normal men. CONCLUSION: The common CFTR mutations were detected in 9/53(17%) infertile men with non-CAVD obstructive azoospermia. Pre-treatment CFTR mutation analysis remains critical to distinguish cystic fibrosis (CF) genotypes for men with non CAVD obstructive azoospermia.