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Introduction: Background: Neff regulatory protein as well as structural proteins such as MPER and V3 of HIV-1 is important targets in vaccine studies. Therefore, in the current study, the Nef-MPER-V3 fusion protein was cloned and expressed in prokaryotic expression system that will be evaluated as a candidate for future vaccine studies. Materials and Methods: The Nef-MPER-V3 sequence was synthesized and inserted into pET-28a(+) vector using Not I/HindIII enzymes. Then, the recombinant construct was expressed in BL21 (DE3) strain of E.coli. For optimization of protein expression, several factors such as time of induction, IPTG concentration and optical density (OD) were evaluated. The recombinant protein was purified on a Ni-NTA agarose column and analyzed by 12% SDS-PAGE. Finally, the fusion protein was confirmed by western blotting. Results: The target gene was successfully cloned into the recombinant vector. The best condition for the expression such as; 2 m IPTG, OD600 = 0.7 and growth 5 hours post-induction were determined and the recombinant protein was purified by affinity chromatography in denaturing condition with urea at a concentration of about 300 μg/ ml. The purified fusion protein migrated as a clear band of around 35 kDa in SDS-PAGE and was detectable using anti-Neff antibody in western blotting. Conclusion: Our results showed that the Nef-MPER-V3 protein was successfully expressed in prokaryotic system and purified protein may provide the antigen for future experiments for immunogenicity evaluation in mice is currently undertaken. [ABSTRACT FROM AUTHOR]

Background: SLC26A4 gene mutations are the second identifiable genetic cause of autosomal recessive non-syndromic hearing loss (ARNSHL) after GJB2 mutations. In this study, the identity and informativeness of D7S2459 marker in SLC26A4 gene region was examined in five ethnic groups of the Iranian population. Materials and Methods: The study was accomplished by genotyping the locus in 165 individuals of five different ethnic groups including Fars, Azari, Torkaman, Gilak and Arab using polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and fluorescent capillary electrophoresis. In this study, results were analyzed by GeneMarker HID Human STR Identity software, GenePop program and Microsatellite Tools software. Results: Analysis of the allelic frequency revealed the presence of 8 alleles of D7S2459 marker in the Iranian population. Among all, allele 148bp at the D7S2459 locus was the most frequent with 31% frequency. The most heterozygosity observed in Azari ethnic by 84.8%. Finally, analysis of PIC value revealed that D7S2459 marker could be considered as a highly informative marker in each ethnic of the Iranian population (PIC value above 0.7). Conclusion: Our data suggested that D7S2459 could be introduced as a highly informative marker in molecular diagnosis of SLC26A4 based ARNSHL by Linkage analysis. [ABSTRACT FROM AUTHOR]

Background & Aims: Iron deficiency anemia is the most common anemia worldwide with significant clinical consequences. The disease is caused by not having enough iron to produce hemoglobin for reasons such as dietary iron deficiency, insufficient iron absorption, chronic bleeding, and genetic factors. Hepcidin protein is the main regulator of systemic iron homeostasis hormone ferroportin. The role of hepcidin is to control the expression of iron-releasing ferroportin levels in duodenal cells and macrophages, modulating iron uptake and recycling. Hepcidin inactivation causes severe iron overload, while increased hepcidin causes anemia. Several factors regulate hepcidin synthesis, including matriptase-2, which reduces hepcidin synthesis. Matryptase-2 is a type 2 membrane serum proteinase, encoded by the TMPRSS 6 gene. This gene is located on the long arm of chromosome 22 and is mainly expressed in the liver. In humans, inactivation of this protein leads to a rare autosomal recessive disease called IRIDA. Several genetic polymorphisms related to iron status have been identified using genomerelated studies. The aim of this study was to investigate the polymorphism of TMPRSS6rs2111833 in patients with iron deficiency anemia in the west of the country. Methods: The present study is a case-control study. Using the sample size determination method available, sampling was performed at a time interval of 3 months with the cooperation of private laboratories in Sanandaj, Dehgolan and Kermanshah. A total of 91 people were randomly included with respect personal consent. Of these, according to the results of ferritin, hemoglobin, red blood cells, white blood cells and platelets 43(47.25%) with iron deficiency anemia and 48(52.75%) healthy controls were enrolled. Five ml of blood sample was collected from all participants and placed in CBC tubes containing anticoagulant and after inverting several times, it was placed in freezer -20. DNA extraction was performed using the extraction kit of Kwsar Company and according to the kit instructions. The quantity and quality of the extracted DNAs were determined using light absorption in spectrophotometer and electrophoresis on 1% agarose gel. The isolated DNA was stored in separate microtubes at -20 ° C until PCR. Then the genotype of individuals was obtained using ARMS-PCR technique and primer sequences used in previous studies. Amplification of the desired fragment was performed using Sinagene Company PCR kit by Rotor-Gene Q (Corbett Life Science) cyclotron device according to the protocol and the PCR product was loaded on 1.8% agarose gel and the quantity and quality were determined. Finally, a statistical test was performed to compare the frequency of genotypes in all samples using Windows SPSS tool and the value of p <0.05 was statistically described. Results: The results showed that the frequency of GG, AG and AA in all participants were 45%, 22% and 33%, respectively, and so for sick and healthy individuals were 47 and 44, 23 and 21 and 30 and 35, respectively. Also, out of 200 alleles, 120(56%) and 80(44%) alleles were G and A, respectively. The portion of G allele in patients and controls group was 58 and 54% and the share of A allele was 42 and 46%, respectively. Hardy Weinberg and heterozygosity equilibria were also examined for the two groups. The Hardy-Weinberg test showed that the assessed population was balanced. Heterozygosity for a gene locus is defined as the frequency of heterozygous individuals for that locus relative to the total population. A gene locus is polymorphic if its heterozygosity is greater than 0.1, and if it is greater than 0.7, it is highly polymorphic. Based on the results of this study, it was seen that the difference between observed and expected heterozygosity in the studied polymorphism is less than 0.1, so the gene position is not polymorphic. Also, co-dominant, dominant, recessive and super-dominant models were examined and in all cases, the Pv value was greater than 0.05. On the other hand, Sixty-nine percent of participants had normal hemoglobin, 41% normal iron, 51% normal ferritin, 83% normal red blood cells, 92% normal platelets and 82% normal white blood cells. On the other hand, 22% of participants had abnormal hemoglobin, 65% abnormal iron, 54% abnormal ferritin, 22% abnormal red blood cells, 2% abnormal platelets and 26% abnormal white blood cells. rs 2111833 showed no significant association with decreased serum hemoglobin, serum iron, serum ferritin, platelets, red and white blood cells. Conclusion: This results showed that the rs2111833 polymorphism were not significantly associated iron deficiency anemia in the studied population. In addition, no significant association was found between rs2111833 and hemoglobin, iron, ferritin, platelets, RBCs and WBCs clinical parameters. Beutler and colleagues investigated the role of TMPRSS6 gene polymorphisms in adults with iron deficiency anemia. Their observations highlighted the role of matriptase-2 in controlling iron metabolism and erythrocyte parameters. Sylvester et al. Reported the association of TMPRSS6 mutations with iron deficiency anemia. Previously, Feinberg et al. reported several mononucleotide polymorphisms associated with iron deficiency anemia, including rs 869320724, rs 767094129, rs 786205059, rs 137853120 and rs 137853119.7 Several studies have shown that the polymorphism rs 2111833 is associated with decreased iron and hemoglobin levels therefore, it related with an increased risk in iron deficiency anemia. In 2015, Gichohi-Wainaina et al. Identified some changes in the partial frequencies of the rs 2111833 alleles in Asian populations compared to the Caucasian population, which matched our findings. Anemia remains a widespread and significant global health problem that needs to be adequately addressed. Although iron deficiency anemia is the leading cause of anemia in most areas, recent research shows that the cause of anemia is complex and specific to each region. To better understand how to help the underlying causes of anemia, including iron deficiency anemia and other nutritional deficiencies, diseases, and hemoglobin disorders, efforts need to be made to make appropriate interventions work under certain conditions. Further studies on a larger patient scale are also necessary to identify potential haplotypes and polymorphisms responsible for the low response to oral iron therapy and may be useful for planning a proper iron supplementation. There were some limitations to this study, such as an insufficient total number of participants. However, these results could enhance our understanding of the role of genetics, particularly single nucleotide polymorphisms, in increasing susceptibility to a variety of diseases. [ABSTRACT FROM AUTHOR]

Background and Objectives CD40L is a membrane protein and a member of the tumor necrosis factor super family (TNFSF) that plays an important role in transferring cell signaling in innate and adaptive immunity. Since this protein plays its role through clustering the receptors on the target cell surface, it seems that the multimeric form of this molecule can have a stronger effect than the natural trimeric form. So in this study we describe cloning and expression of dodecameric soluble CD40L through self multimerizing protein, surfactant protein D (SP-D), in the HEK293 cell line. Materials and Methods In this experimental study, the SPD-CD40L was designed in silico and cloned in pcDNA3.1(+) plasmid. HEK293 cells were transfected and used as the expression host cells. The expression of SPD-CD40L was determined at the RNA level by RT-PCR method. After the purification of the recombinant proteins by affinity chromatography, the molecular weight of the recombinant protein was determined by SDS-PAGE. To evaluate the specificity of protein, ELISA and Dot Blot were used. Results The results indicated that HEK293 cells were transfected and recombinant protein was expressed 24 and 48 hours after Transfection. Moreover, the results of ELISA and Dot Blot methods represented the specificity of recombinant SPD-CD40L chimeric protein. Conclusions Chimeric protein SPD-CD40L was expressed successfully by pcDNA3.1(+) in eukaryotic HEK293 cell line and the molecular weight of 4-trimeric (dodecameric) protein was shown to be consistent with the expected amount. [ABSTRACT FROM AUTHOR]