In silico analysis of Brucella abortus Omp2b and in vitro expression of SOmp2b

PURPOSE: At present, there is no vaccine available for the prevention of human brucellosis. Brucella outer membrane protein 2b (Omp2b) is a 36 kD porin existed in common Brucella pathogens and it is considered as priority antigen for designing a new subunit vaccine. MATERIALS AND METHODS: In the current study, we aimed to predict and analyze the secondary and tertiary structures of the Brucella abortus Omp2b protein, and to predict T-cell and B-cell epitopes with the help of bioinformatics tools. Subsequently, cloning and expression of the short form of Omp2b (SOmp2b) was performed using pET28a expression vector and Escherichia coli BL21 host, respectively. The recombinant SOmp2b (rSOmp2b) was purified with Ni-NTA column. RESULTS: The recombinant protein was successfully expressed in E. coli host and purified under denaturation conditions. The yield of the purified rSOmp2b was estimated by Bradford method and found to be 220 microg/mL of the culture. CONCLUSION: Our results indicate that Omp2b protein has a potential to induce both B-cell- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

Effect of poly lactic-co-glycolic acid-ibuprofen on ICAM-1 and VCAM-1 expression in a mice adhesion model

Background: in this study, we compared the effect of ibuprofen [IB] while incorporating by Poly Lactic-co-Glycolic Acid [PLGA] nanofiber on expression of adhesion molecules ICAM-1 and VCAM-1 in a mice adhesion model

Materials and Methods: using an adhesion model were induced in mice, PLGA-IB and PLGA membranes and IB were sutured between the abdominal wall and peritoneum after surgical operation to reveal the best membrane for prevention of postoperative adhesion bands by comparison of ICAM-1 and VCAM-1 expression

Results: compared with other groups, PLGA-IB showed a greater ability to reduce ICAM-1 and VCAM-1 expression

Conclusion: these results suggested that in considering the FDA approved polymers, PLGA-IB could be introduced as a potential candidate for prevention of abdominal post-surgery inflammation and adhesion band formation after surgeries

Higher expression level and lower toxicity of genetically spliced rotavirus NSP4 in comparison to the full-length protein in E. coli

Background: Rotavirus group A [RVA] is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 [NSP4] is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals

Objectives: The aim of this study is the evaluation of rotavirus ANSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein

Materials and Methods: Splicing by overlap extension [SOEing] PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length [FL-NSP4] and the spliced [S-NSP4] cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 [DE3] by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test

Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable

Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain [S-NSP4]. SNSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future

Ineffectiveness of methylation in rgulation of VHL, ECAD, and RUNX3 genes in erythroid cells differentiated by erythropoietin

Vast variety of intermediate factors including cell cycle regulators, growth factors, transcription factors, and signaling pathways are involved in hematopoietic stem cell [HSC] commitment and differentiation into distinct lineages. VHL, Ecad, and RUNX3 are among these. Epigenetics is currently introduced as a potential mechanism to control the gene regulation. The aim of this study is to reveal the correlation between the expression level and methylation pattern of mentioned genes after in vitro differentiation of cord blood HSCs into erythroid lineage mediated by erythropoietin. After isolation and expansion, the CD34+ cord blood stem cells were divided into two parts. The first part was used to extract the DNA and RNA and the second to differentiate into erythroid lineage. Methylation specific PCR [MSP] and Real-time PCR were used to determine the methylation status and expression levels of the genes, respectively. Although the significant upregulation observed for VHL and Ecad genes and a down-regulation for RUNX3 gene after differentiation, no remarkable changes were seen in methylation pattern compared with cord blood HSCs by MSP technique. It is appearing that methylation pattern in promoter region has not an effective role in expression of VHL, Ecad, and RUNX3. Moreover, considering the inability of MSP method to detect subtle differences in methylation level a more sensitive method is needed to distinguish the methylation levels of these genes before and after erythroid differentiation

Correlation between methylation and expression level of P15 and P16 genes during differentiation of cord blood stem cells into erythroid lineage mediated by erythropoietin

Several influential factors such as transcription factors and intracellular signaling components are involved in differentiation of stem cells into a specific lineage. P15 and p16 proteins are among these factors. Accumulating evidences has introduced the epigenetic as a master regulator of these factors during lineage specification. The main objective of this study is to determine the correlation between the expression level and methylation pattern of P15 and P16 genes in erythroid lineage after in vitro differentiation by erythropoietin [EPO]. The purified and expanded CD34+ cord blood stem cells were differentiated into erythroid lineage in the presence of EPO. DNA was isolated from both cord blood stem cells and differentiated cells. The Real-Time PCR performed using cDNA and the isolated DNA was used in methylation Specific PCR [MSP] reaction for methylation pattern analysis in both pre and post differentiation stages. The study demonstrated that P15 and P16 genes have partial methylation after erythroid differentiation by EPO. The Expression of P15 gene was higher after differentiation and the expression of P16 gene had a slightly decreased level in post differentiation stage. Significant increase in P15 gene expression after differentiation to erythroid lineage, suggests the remarkable efficacy of this gene in erythroid function. According to upregulation of P15 gene after differentiation despite unchanged methylation status and slight down regulation of P16 gene with slight hyper-methylation of the gene it can be suggested that although the methylation can affects the expression level of P16 gene, the P15 gene is not affected by this mechanism during erythroid differentiation mediated by EPO

[Serum zinc concentrations in children with acute bloody and watery Diarrhoea]

Objectives: The role of zinc in the pathogenesis of diarrhoea is controversial. This study was conducted to compare serum zinc levels in children with acute diarrhoea to those found in healthy children

Methods: This case-control study was carried out at the Qazvin Children's Hospital in Qazvin, Iran, between July 2012 and January 2013. A total of 60 children with acute diarrhoea [12 children with bloody diarrhoea and 48 children with watery diarrhoea] and 60 healthy children were included. Zinc levels for all subjects were measured using a flame atomic absorption spectrophotometer and data were analysed and compared between groups

Results: Mean serum zinc levels in the patients with acute bloody diarrhoea, acute watery diarrhoea and the control group were 74.1 +/- 23.7 microg/dL, 169.4 +/- 62.7 microg/dL and 190.1 +/- 18.0 microg/dL, respectively [P = 0.01]. Hypozincaemia was observed in 50.0% of children with acute bloody diarrhoea and 12.5% of those with acute watery diarrhoea. None of the patients in the control group had hypozincaemia [P = 0.01]

Conclusion: Children with acute bloody diarrhoea had significantly reduced serum zinc levels in comparison to healthy children. However, a study with a larger sample size is needed to examine the significance of this trend

763C>G polymorphism of the secretory PLA2IIa gene is associated with endometriosis in Iranian women

Endometriosis is a chronic gynecological disease resulting from complex interactions between genetic, hormonal, environmental and oxidative stress and intrinsic inflammatory components. The aim of this study was to investigate the potential association of the 763C>G polymorphism in the secretory phospholipase A2 group IIa gene [PLA2G2A] with the risk of endometriosis in Iranian women. Ninety seven patients with endometriosis along with 107 women who were negative for endometriosis after laparoscopy and laparatomy, and served as the control group, were enrolled for this cross-sectional study. Samples were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method. Multivariate analysis was used to examine the association between the risk of endometriosis and the 763C>G polymorphism of PLA2G2A. Genotype distributions of PLA2G2A were significantly different between patients and the controls [p<0.001, OR=0.22, 95% CI=0.21-0.39]. Correlation analysis showed that there was a significant association between the normal homozygous genotype and susceptibility to endometriosis [p<0.001]. The present study suggests that the 763C>G polymorphism of PLA2G2A plays an important role as an independent factor in the risk of endometriosis in Iranian women

Challenges in the treatment of Iranian patients with leukemia in comparison with developed countries from the perspective of specialists

Evaluation of the factors associated with treatment process of leukemia and comparison with current related approaches in developed countries can present a good indicator to assess the weak and strong points in healthcare system of our country in leukemia treatment. The objective of this research is general and specific description of the challenges and shortcomings in Iranian healthcare system and monitoring of hematologic malignancies as well as comparison with developed countries. Our study is a descriptive-cross-sectional study. 100 hemato-oncologist , pathologists, and faculty members throughout the country were selected by random cluster sampling. Data collected using questionnaires with Cronbach's alpha coefficient of 0.76 . SPSS and Chi-square test were used for data analysis. According to the specialists, lack of advanced diagnostic facilities as well as cell and BM banks together with high treatment expenses are the main factors contributing to poor treatment processes in Iran, which are far from worldwide standards. The use of novel currently methods used in developed countries for leukemia treatment, financial and psychological support of patients under treatment , making underprivileged provinces well-equipped ,balanced specialist service distribution relative to capital city either in diagnosis or treatment are factors which makes system standardized. Moreover, integrated institutional work in relation to leukemia incidence and statistical analysis of mortality and morbidity rate can pave the way for reducing and eliminating the problems in diagnosis and treatment of leukemia patients

Correlation between PPAR gamma protein expression level in granulosa cells and pregnancy rate in IVF program

Peroxisome proliferative-activated receptors [PPARs] are nuclear receptors that involved in cellular lipid metabolism and differentiation. The subtype gamma of the PPAR family [PPAR gamma] plays important roles in physiologic functions of ovaries. To determine correlation between PPAR gamma protein level in granulosa cells and pregnancy rate in women undergoing in-vitro fertilization [IVF] treatment. In this cross-sectional study, twenty-five samples of granulosa cells were collected from women referred to an IVF treatment center. PPAR gamma protein expression level in granulosa cells was determined in comparison with beta -actin level as control gene with Western blot test. Laboratory pregnancy was determined by a rise in blood beta -hCG level fourteen days after embryo transfer. Correlation analyses were used to test for associations between the oocytes and pregnancy occurrence as outcome variables and PPAR gamma protein expression level. Correlation analysis indicated that there was no significant relationship between granulosa cells PPAR gamma protein level with IVF parameters including number of matured oocytes and the ratio of fertilized to matured oocytes. Comparison of granulosa cells PPAR gamma protein level with positive and negative laboratory pregnancy revealed also no significant relationship. According to the results of this study, PPAR gamma protein level in granulosa cells could not be directly correlated to the success rate of IVF