Avicenna Journal of Medical Biotechnology
Year:2018 | Volume:10 | Issue:1|Papers Count:10

Alzheimer’s Disease (AD), the leading cause of dementia worldwide, is an irreversible progressive neurodegenerative disorder characterized by cognitive impairment and functional disability. Devastating nature of AD leads to serious social and economic impacts on the healthcare systems which implies the necessity of its proper management. It has been demonstrated that patients’ quality of life and their overall prognosis has a significant negative correlation with the severity of AD. ...

Background: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, GRA7 plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice.Methods: In this study, 733 bp of GRA7 gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containingGRA7 and ROP2 genes were administered via IMaccording to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis.Results: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-γ levels were significantly higher than controls (p<0.05), whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group (p<0.01). In contrast, IgG1 antibodies were similar between the two groups (p>0.5). So, survival time in the immune groups was significantly prolonged in comparison to control ones (p<0.01).Conclusion: The immunized mice by DNA vaccine produce higher titration of IFNγ, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktailGRA7/ROP2 is a potential vaccine candidate against toxoplasmosis.

Background: Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding sequence.Methods: The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature peptide encoding the traceable avimer molecule was carried out in Escherichia colistrain BL21 using IPTG (Isopropyl b-D-1-thiogalactopyranoside) induction process.The expression level of the 11kDa traceable avimer was studied by SDS-PAGE, western blot and ELISA analysis.Results: Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11kDa C426 avimer molecule was detectable without any degradation compared with the control group.Conclusion: Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity and traceability features.

Background: b-Cyclodextrin functionalized hyper-branched polyglycerol (HBCD: b-CD-g-PG), a biocompatible polymer, has recently been proposed for delivery of poorly water soluble compounds.Methods: The present study examines the interaction of HBCD with RNA, utilizing a constant concentration of RNA and different HBCD/RNA ratios of 1.16 to 1.1, at physiological condition in an aqueous solution. Circular Dichroism (CD), UV-visible, FTIR spectroscopic methods, zeta potential and Dynamic Light Scattering (DLS) were used to analyze the particle formation, particle charge, particle size, aggregation, RNA conformation, binding constant and mode, and the effect of polymer complexation on RNA stability.Results: The results indicate that the interaction of RNA with HBCD leads to the formation of a linear dendritic supramolecule biopolymer with an overall binding constant of KHBCD/RNA=1.25 ´ 103.Conclusion: The small sized synthesized polymer can be considered as an appropriate system for preventing RNA aggregation and protecting the gene by host-guest interaction.

Background: Hydro-distillation (HD) method is a traditional technique which is used in most industrial companies. Microwave-assisted Hydro-distillation (MAHD) is an advanced HD technique utilizing a microwave oven in the extraction process.Methods: In this research, MAHD of essential oils from the aerial parts (leaves) of rosemary (Rosmarinus officinalis L.) was studied and the results were compared with those of the conventional HD in terms of extraction time, extraction efficiency, chemical composition, quality of the essential oils and cost of the operation.Results: Microwave hydro-distillation was superior in terms of saving energy and extraction time (30min, compared to 90 min in HD). Chromatography was used for quantity analysis of the essential oils composition. Quality of essential oil improved in MAHD method due to an increase of 17% in oxygenated compounds.Conclusion: Consequently, microwave hydro-distillation can be used as a substitute of traditional hydro-distillation.

Background: MicroRNAs (miRNAs) are implicated in various biological processes including anticoagulation. However, the modulation of miRNA by pharmacological intervention such as warfarin treatment in patients receiving warfarin has not been disclosed yet. The aim of this study work was to assess the effect of warfarin drug on expression level of mir-133a-3p in patients with mechanical heart valve replacement.Methods: In this research, the expression level of miRNA-133a-3p was analyzed in Peripheral Blood Mononuclear Cells (PBMCs) from mechanical valve replacement patients who had received warfarin for at least 3 months continuously. Quantitative RT-PCR method was used for this assay.Results: Our findings indicated a significant diffrence between the rate of miR-133a-3p expression in individuals receiving warfarin and the control group (p<0.01). There was also a statistically significant difference in miR-133a-3p expression in patients with different ages (p<0.05) suggesting that the rate of miR-133a-3p expression in persons receiving warfarin is related to age. However, other variables like warfarin dose, International Normalized Ratio (INR), gender, and Body Mass Index (BMI) were not significantly effective on the miR-133a-3p experssion rate in individuals receving warfarin.Conclusion: Based on our results, it can be concluded that miR-133a-3p is involved in the coagulation pathway. The recent result indicates that warfarin affects the expression of miR-133a. This expression may be potentially important for treatment by anticoagulants. Awareness of the time course of miRNA expression profile can improve efficiency of response to warfarin.

Background: Type 4 pili (T4P) is an important virulence factor of Pseudomonas aeruginosa (P. aeruginosa). T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ380-706) was produced as a recombinant protein.Methods: A 981 bp fragment of C-terminal of the pilQ secretin (pilQ1138-2118) from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ1138-2118 gene in the recombinant construct (pET28a/pilQ) was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ380-706 confirmed by Western blot analysis and twitching inhibition assay.Results: The PCR and enzymatic digestion results showed the presence of the pilQ1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ380-706 is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ380-706 protein. The PilQ380-706 protein showed high biological activity in all of these standard assays.Conclusion: Since, the PilQ380-706 protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.

Background: Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds.Methods: In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head de condensation was applied for the second and third experiments. In experiment 2, in vitro matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm.Results: The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control.Conclusion: In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+ GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control.

Background: Riboflavin (vitamin B2) is an essential component of the basic metabolism, and an important nutritional and growth factor in humans, animals, plants and micro-organisms. It has been widely used in the fields of pharmaceuticals, feed and food additives. The industrial production of riboflavin mostly relies on the microbial fermentation. Designing an appropriate fermentation medium is of crucial importance to improve the riboflavin production.Methods: In this study, sequential methodology combining a screening test of minerals by Plackett-Burman (PB) and an optimization test by Central Composite Design (CCD) was applied to enhance riboflavin production byBacillus subtilis ATCC 6051 in shake flasks.Results: Initially, one-factor-at-a-time approach was applied to evaluate the effect of different carbon sources. The results showed that fructose was significantly most effective on biomass and riboflavin production. After that, 13 minerals [CaCl2, CuCl, FeCl3, FeSO4, AlCl3, Na3MoO4, Co (NO3) 2, NaCl, KH2PO4, K2HPO4, MgSO4, ZnSO4, and MnSO4] were studied with the screening test. The results revealed that concentration of MgSO4, K2HPO4, and FeSO4 had greater influence on riboflavin production (p< 0.05). A CCD with five factors (concentration of fructose, MgSO4, K2HPO4, FeSO4, and yeast extract) at five levels was then used to determine the maximum riboflavin concentration. The optimal concentrations (g/l) of these variables determined by Response Surface Methodology (RSM) were fructose, 38.10; MgSO4, 0.85; K2HPO4, 2.27; FeSO4, 0.02; and yeast extract, 4.37.Conclusion: Statistical experimental design offers a practicable approach to the implementation of medium optimization. From an industrial view point, our optimum medium, besides fructose and a small amount of yeast extract, is mainly composed of common and cheap inorganic salts, which are available to the industrial riboflavin production.

Background: Recurrent Aphthous Stomatitis (RAS) is one of the most common diseases of the oral cavity all over the world (5-66%). RAS has a multifactorial etiology, while psychological factors such as stress and anger play a role in its manifestation. The serotonergic mechanisms particularly the serotonin-transporter gene (5-HTT) may affect the risk of psychological alterations and stress response. The aim of the present study was to evaluate the polymorphism of the promoter region of5-HTT (5-HTTLPR) in the patients with RAS, compared to that in the control subjects.Methods: In this case-control study, 100 patients with RAS and 100 healthy subjects were enrolled. PCR was performed on DNA of the samples, using a pair of primers capable of distinguishing S/L alleles and replicating5-HTTLPR.Results: No statistically significant difference existed between LL and LS genotype frequencies in the case and control groups. However, SS genotype frequency was significantly higher in the case group, as compared to the control group (p=0.001).Conclusion: The conclusion of the present study demonstrated that S allele could approximately double the risk of RAS.