Cloning, expression, purification and functional analysis of a specific multi-epitope protein from multi drug resistance Acinetobacter baumannii.

BACKGROUND AND OBJECTIVES: Immunization is a promising strategy to combat against the life-threatening infections by Multi Drug Resistance Acinetobacter baumannii. In this study, we directed to design and evaluate the efficacy of a recombinant multi-epitope protein against this pathogen. MATERIALS AND METHODS: Epitopes prediction was performed for candidate proteins OmpA and BAM complex (BamA, BamB, BamC, BamD, BamE) from A. baumannii, using immune-informatics tools with high affinity for the human HLA alleles. After expression and purification of the recombinant protein, its functional activity was confirmed by interaction with positive sera. RESULTS: Cloning and expression of the desired multi-epitopes protein were verified. Circular Dichroism study showed the secondary structure and proper refolding of the recombinant protein was achieved and matched with computational prediction. There was a significant interaction between designed protein with antibodies presented in ICU patients' and staff's sera. CONCLUSION: The interaction of the recombinant protein with patients' sera antibodies suggests that it may be a promising determinant protein for immunization against of MDR A. baumannii.

Induction of Immunogenic Response in BALB/c Mice by Live and Killed Form of Recombinant Lactococcus lactis Displaying EG95 of Echinococcus granulosus.

Background: Cystic echinococcosis is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods: The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results: Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. Mucosal IgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion: Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice.

Adenosine decreases oxidative stress and protects H2O2-treated neural stem cells against apoptosis through decreasing Mst1 expression.

Overproduction of free radicals during oxidative stress induces damage to key biomolecules and activates programed cell death pathways. Neuronal cell death in the nervous system leads to a number of neurodegenerative diseases. The aim of the present study was to evaluate the neuroprotective effect of adenosine on inhibition of apoptosis induced by hydrogen peroxide (H2O2) in bone marrow-derived neural stem cells (B-dNSCs), with focus on its regulatory effect on the expression of mammalian sterile 20-like kinase 1 (Mst1), as a novel proapoptotic kinase. B-dNSCs were exposed to adenosine at different doses (2, 4, 6, 8 and 10 µM) for 48 h followed by 125 µM H2O2 for 30 min. Using MTT, terminal deoxynucleotidyl transferase dUTP nick-end labeling and real-time reverse transcription polymerase chain reaction assays, the effects of adenosine on cell survival, apoptosis and Mst1, nuclear factor (erythroid-derived 2)-like 2 and B-cell lymphoma 2 and adenosine A1 receptor expression were evaluated in pretreated B-dNSCs compared with controls (cells treated with H2O2 only). Firstly, results of the MTT assay indicated 6 µM adenosine to be the most protective dose in terms of promotion of cell viability. Subsequent assays using this dosage indicated that apoptosis rate and Mst1 expression in B-dNSCs pretreated with 6 µM adenosine were significantly decreased compared with the control group. These findings suggest that adenosine protects B-dNSCs against oxidative stress-induced cell death, and therefore, that it may be used to promote the survival rate of B-dNSCs and as a candidate for the treatment of oxidative stress-mediated neurological diseases.

Supplementation of freezing media with stromal cell-derived factor-1α preserves human sperm from cryodamage.

The destructive effects of sperm cryopreservation result in reduced sperm motility and increased apoptosis. Oocytes, endometrium, and follicular fluid express stromal cell-derived factor-1 alpha (SDF-1α) or C-X-C motif chemokine ligand 12 (CXCL12) while its specific receptor chemokine, CXC motif receptor 4 (CXCR4) is expressed in the head of sperm. SDF-1α can increase sperm motility and preserve normal mitochondrial status. The present study intends to investigate whether the addition of SDF-1α to freezing extender can facilitate cryosurvival of spermatozoa and how SDF-1α protects spermatozoa against damages during cryopreservation. In this study, we collected 22 semen samples from healthy donors and treated them with different concentrations of SDF-1α, followed by cryopreservation for one month. We measured sperm motility by CASA, mitochondrial ROS generation by flow cytometry using the probe MitoSOX Red™ (MSR) to measure mitochondrial superoxide anion (O2-•), DNA fragmentation by flow cytometry according to the TUNEL kit, and expressions of Bcl-2 and Bax by RT-qPCR in freeze-thawed sperm. The results showed that SDF-1α attenuated mitochondrial ROS generation at different doses, particularly the 250 ng/ml treated samples which, in turn, reduced the expressions of pro-apoptotic genes such as Bax. Eventually, SDF-1α reduced DNA fragmentation and ameliorated sperm motility in the 1-100 ng/ml treated samples during cryopreservation. The present study, for the first time, demonstrated that SDF-1α dose-dependently moderated oxidative stress injury in human sperm by reduction of mitochondrial ROS generation. It could subsequently cause a decrease in apoptosis during freeze-thawing and protect human spermatozoa from cryodamage.

The relationship between vitamin D receptor (VDR) polymorphism and the occurrence of osteoporosis in menopausal Iranian women.

BACKGROUND: Osteoporosis, a multifactorial disease with reduced bone mineral density which increases the probability of bone fractures, is caused by calcium deficiency, and its incidence increases with age. It has been determined that mutations in functional regions of vitamin D receptor gene will affect the metabolism of minerals especially calcium and, therefore, bone density. The present study evaluates the relation between vitamin D receptor polymorphisms, TaqI (rs731236) and ApaI (rs7975232), and osteoporosis in menopausal Azari women in Zanjan province. MATERIALS AND METHODS: This case-control study has been conducted on 50 menopausal women suffering from osteoporosis and 50 menopausal women who did not suffer from osteoporosis in Zanjan province. The diagnosis of osteoporosis was confirmed using DEXA instrument. Peripheral blood was collected from the subjects and controls to extract DNA and assess the ApaI and TaqI polymorphisms using PCR-RFLP method. The results were interpreted using independent T-test, chi-square, and Pearson correlation coefficient with a p-value less than 0.05. RESULTS: There was not a significant difference between the frequency of ApaI (AA/Aa/aa) and TaqI (TT/Tt/tt) genotypes in cases (mean age 68.72) and controls (mean age 64.7) (p=0.37 and p=0.64, respectively). In addition, ApaI/TaqI allele haplotype in osteoporotic population showed non-significant relation (p value=0.563) compared with the control group. DISCUSSION AND CONCLUSION: The relationship between the genotypes and osteoporosis, cancers, and mineral metabolism disorders has been studied for a long time. Although there has been a significant relation between the aforementioned genotypes and osteoporosis or reduced mineral density-related bone fractures in some studied, some other studies have opposing results. Therefore, it is only possible to reach an acceptable conclusion by studying the haplotype of the polymorphisms in subjects.