Iranian Journal of Biotechnology
Year:2017 | Volume:15 | Issue:3|Papers Count:9

Background: Although peripheral nerves show capacity for regeneration after injury to a certain extent, the extent of regeneration is not remarkable. Previous studies have suggested that through the production of growth factors or extracellular matrix components, mesenchymal stem cells may enhance nerve regeneration.Objectives: In the present study, the therapeutic potency of the Bone Marrow Stromal Cells (BMSCs) associated with Poly L-lactic-co-glycolic acid (PLGA) nanofi ber Scaff olds on rat sciatic nerve repair was evaluated.Material and Methods: Thirty adult male Wistar rats (220-250 g) were divided randomly into six groups, including control 1 (transected sciatic nerve), control 2 (transected sciatic nerve and stitched), Sham, PLGA, BMSCs, and PLGA+BMSCs.Functional recovery was evaluated at the end of 2nd, 4th, 6th, and 8th weeks after surgery using sciatic functional index (SFI) and hot water test. After killing all rats at the end of 8th week, their sciatic nerves were removed, fi xed, and processed for the histological examination and analysis by the Motic software.Results: A signifi cant recovery of the sciatic nerve function was observed in the PLGA+BMSCs transplanted group at the 8th week after surgery as demonstrated by SFI and hot water fi ndings. Histological examinations also showed a signifi cant improvement in the PLGA+BMSCs group compared to the control 1, 2, Sham, PLGA and BMSCs groups.Conclusion: BMSCs associated with PLGA nanofi ber scaff old might be useful for improving the functional peripheral nerve repair having some clinical outcome.

Background: Artesunate has recently been used in some pharmacological preparation to induce tumor cell apoptosis. The drug is a semi-synthetic derivative of artemisinin, traditionally used for its antimalarial. However, up to now, its anticancer mechanism against diff erent types of tumors is not known.Objectives: The most important purposes of the present research was fi rstly investigating induction of apoptosis on human breast cancer MCF-7 cells by the drug and, in the second place, introducing its possible mechanism of action.Materials and Methods: The MTT assay was used to investigate the inhibitory eff ect of artesunate on growth of breast cancer MCF-7 cells. For this aim, diff erent concentrations of artesunate were used to treat the cells and fl ow cytometry assay was done followed by annexin V-FITC/PI staining. The activities of caspase-3, -8 and -9 were then determined by relative assay kits.Results: Based on the results from MTT assay, it was found that artesunate could signifi cantly inhibit the growth of MCF-7 cells in a dose- and time-dependent manner. On the other hand, the fl ow cytometry fi ndings showed that the anti-proliferative activity of artesunate on MCF-7 cells is due to apoptosis. Besides, caspase colorimetric assays revealed a signifi cant rise in cellular levels of the initiators (caspase-8 and -9) and eff ector (caspase-3) in the cells treated by artesunate.Conclusions: According to our results, it could be concluded that artesunate could inhibit the growth of MCF-7 breast cancer cells through induction of apoptosis by intrinsic and extrinsic caspase-dependent pathways. Therefore, we claim that artesunate could be introduced as a suitable candidate for the treatment of the breast cancer.

Background: T cell Immunoglobulin, Mucin (TIM) -3, is a type I trans membrane glycoprotein belonging to TIM family. This receptor expresses on T helper type 1 (Th1) cells that binds to galectin-9 (Gal9); inducing an inhibitory signal. As a result, apoptosis of Th1 cells occurs and cytotoxicity of CD8 T cells becomes evident in vitro. Therefore, this immunomodulatory molecule may be used as a novel target for clinical purposes. The production of camel polyclonal antibodies against TIM-3-expressing cell line was the purpose of this study.Objectives: In this study, we aimed to use HEK 293 cells expressing human TIM-3 to obtain camel polyclonal antibody against TIM-3 by immunization.Materials and Methods: A pre-synthesized human TIM-3cDNA was inserted into pcDNA3.1 plasmid and the new construct was transfected in HEK cell. TIM-3 expression was confi rmed by qRT-PCR and fl ow cytometry. A camel (6 months old) was immunized with the lysate prepared from rTIM-3 expressing HEK cells 4 times. The anti-TIM-3 antibody level was evaluated using ELISA method.Results: TIM-3 was successfully cloned in HEK cells with 88% success rate. High level of anti-TIM-3 antibody was detected in the serum of the camel immunized with the recombinant cell lysate, after fi nal injection.Conclusions: Our rhTIM-3 cell display system can be useful for future diagnostic or therapeutic approaches.

Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein.Objectives: Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system.Materials and Methods: Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred intoE. coli DH10Bac competent cells. Site-specifi c transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confi rmed via PCR using specifi c primers and PUC/M13 universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fl uorescence microscopy and western analysis, respectively.Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verifi ed. Accuracy of transfection process was confi rmed by GFP fl uorescence microscopy.VP2 expression was verifi ed by SDS-PAGE and western analysis.Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.

Background: Bioactive compounds such as terpenoids, chondroitin sulfate, and polysaccharides with added value can be found in prestine marine creatures. These compounds often do have highly valuable therapeutic applications such as being antioxidant, antitumoro genic, anti-infl ammatory and anti-angiogenic. For the latter, varieties of angiogenesis factors can suppress this issue within the bodily tissues.Objectives: The anti-angiogenic and anti-metastatic capacity of a polysaccharide derived from brittle star was investigated.Material and Methods: The anti-proliferative eff ect of derived polysaccharide on umbilical vein endothelial cells (HUVEC) was measured using MTT (dimethyl thiazol-2-yl] -2, 5-diphenyl tetrazolium bromide) assay. The anti-angiogenic eff ect of the isolated polysaccharide was examined by Chorioallantoic membrane (CAM) assay. The transcriptional expression of VEGF (Vascular Endothelial Growth Factor) was evaluated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The anti-metastatic activity was investigated via scratch-wound healing assay. The levels of Paxillin and Matrix Metalloproteinase-9 (MMP-9) expression were analyzed by RT-PCR. Statistical analysis and mean comparisons (p<0.05) were carried out by SPSS 16.Results: Our results elucidated that the brittle star isolated polysaccharide exerted a dose dependent cytotoxic eff ect on the HUVEC endothelial cells. The CAM assay exhibited potent anti-angiogenic activityin vivo. The RT-PCR analysis showed that the extracted polysaccharide (40, 60 mg.mL-1) down-regulated the VEGF expression. Further, the diminished attachment of endothelial cells demonstrated that the anti-invasiveness of the derived polysaccharide (25, 50 mg.mL-1) was administrated via down-regulation of paxillin and MMP-9 mRNA expression.Conclusions: Taken together, these results indicated that the polysaccharide extracted from brittle star was able to decrease the viability of the HUVEC cells, to suppress angiogenesis, and possibly act as a natural anti-angiogenic and anti-metastatic marine organic compound against angiogenesis related pathologies.

Background: Diagnostic molecular marker studies are in vogue to have insight of most prevalent animal diseases including cancer.Objectives: Gene expression profi ling of pro and anti-apoptotic genes was conducted in dog Lymphoma, CTVT, SCC, granuloma, perianal adenocarcinoma and mammary tumors.Materials and Methods: Cancerous tissues of 21 aff ected animals were obtained. Total RNA was extracted followed by cDNA synthesis. Comparative Ct method viaTaqman assay (RT-qPCR) was used to quantify corresponding mRNA molecules, Tp53 and Hspb1, as normalized by GAPDH as the reference gene.Results: Hspb1 showed ectopic expression in lymphoma, CTVT and mammary tumors; its down-regulation was observed in granuloma and oral SCC with fold diff erence (FD) of ±35. Similarly, Tp53 as the tumor suppressor gene with proapoptotic properties, showed up-regulation in all tumor types, notably 80% of mammary tumors and 60% of CTVT. The FD values were 33.31 and 2.27, respectively.Conclusion: Altered transcriptomic response of Hspb1 and Tp53 was observed in all cancer types of Canis familiaris. The resulting profi le depicts the involvement of the genes in cancer pathways. Thus, the data might be helpful for diagnosis, prognosis, identifi cation and classifi cation of these widespread neoplasms in this species.

Background: Pseudomonas protein expression in E. coli is known to be a setback due to signifi cant genetic variation and absence of several genetic elements inE. coli for regulation and activation of Pseudomonas proteins. Modifi cations in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli.Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonassp. and E. coli.Materials and Methods: Pseudomonas-Escherichia shuttle expression vectors, pCon2 (3), pCon2 (3) -Kan and pCon2 (3) -Zeo as well asE. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαAbased vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastino lytic activity assay.Results: The integration of two series of IPTG inducible expression cassettes in pCon2 (3), pCon2 (3) -Kan and pCon2 (3) -Zeo, each carrying anE. coli lac-operon based promoter, Plac, and a tightly regulated T7 (A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastino lytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2 (3) -derived vectors harboring expression cassettes controlled byPT7 (A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 andO4, and lacIq.Conclusions: The constructs off ered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. andE. coli for downstream applications such as in industries and structural biology study.

Background: Canola is an agro-economically oilseed crop. Yield loss due to fungal disease of stem rot caused by Sclerotinia sclerotiorumis a serious problem in canola cultivation. Thaumatin-like proteins are large groups of the pathogenesis-related proteins which provide resistance to the fungal infection in response to invading pathogens and play a key role in plant defense system.Objectives: Transformation of the rice tlp into canola via Agrobacterium -mediated transformation and evaluation of the antifungal activity of the expressed TLP in the transgenic events on theS. sclerotiorum growth was subject to investigation.Materials and methods: The canola (R line Hyola308) was used for transformation experiment. The vector, pBITLPRA1, was used for the stable transformation. The PCR and southern blotting techniques were used to confi rm transgene’s presence in the transgenic canola events. Antifungal activity of transgenic plants was evaluated by the radial diff usion and spore germination assays. T2 transgenic plants were evaluated by the intact leaf inoculation method in greenhouse assay.Results: In this study, pBITLPRA1 construct containing tlp gene was introduced into canola and the transformed plants were verifi ed by PCR. The glucanase activity oftlp gene in T0 generation was measured and transgenic plants with high activity were assessed by Southern blot analysis to confi rm the copy number of the gene. Also, antifungal activity of the single copy T0 transgenic plants against Sclerotinia sclerotiorum was evaluated by radial diff usion and spore germination assays. In greenhouse assay, evaluation of T2 transgenic plants by the intact leaf inoculation method demonstrated that following the infection withS. sclerotiorum, there was a signifi cant reduction in the lesion’s diameter in transgenic lines compared to the non-transgenic ones.Conclusions: These results revealed that expression of TLP has an inhibitory eff ect against fungus compared to nontransgenic plants bothin vitro and in vivo (i.e., greenhouse condition). These transgenic lines could be used as the additional sources of disease resistance for canola breeding program.

Background: The trans version of G to T (G894T) in human endothelial nitric oxide synthase (eNOS) gene has profound eff ects such as male infertility, recurrent miscarriage, multiple sclerosis and cardiovascular diseases.Objectives: Development of a new Multiplex Tetra-Primer Amplifi cation Refractory Mutation System - Polymerase Chain Reaction (T-ARMS-PCR) for detection of rs1799983 (G894T) in the humaneNOS was sought.Materials and Methods: A T-ARMS-PCR for rs1799983 polymorphism in a single-step PCR was carried out, and the results were confi rmed by PCR-RFLP technique in 82 infertile men with varicocele.Results: The results showed that GG (varicocele infertile men), GT and TT genotypes appear to be 53.65%, 34.14%, and 12.19%, respectively. Full accordance between PCR-RFLP and T-ARMS-PCR methods for genotyping of rs1799983 polymorphism was found.Conclusions: This is the fi rst work that describes a rapid, relatively cheap, high throughput detection of G894T polymorphism ineNOS that can be used in large scale clinical studies.