[Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines].

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were -544.00 and -469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.

Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization.

Angiogenesis is among the most important mechanisms that helps cancer cells to survive, grow and undergo metastasis. Therefore, inhibiting angiogenesis will suppress tumor growth. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are believed to be important players of angiogenesis. The goal of this study was to evaluate the success of a novel nanobody against VEGFR2 in tethering its target inside the endoplasmic reticulum and preventing its transport to the cell membrane. Nanobody sequence was cloned in a mammalian vector in fusion with green fluorescent protein and a KDEL retention signal. After transfection of 293KDR cells with this expression vector, surface localization of VEGFR2 was monitored by flow cytometry. This study demonstrates that our intrananobody is effective in targeting VEGFR2 receptor, and therefore, it is a powerful tool to downregulate a surface-exposed target protein, and in this capacity, it has potential to be used as a therapeutic protein to inhibit growth of tumors.

Molecualr Cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid.

Yersinia pestis which is the causative agent of pneumonic plague and distributed in all continents has led to many deaths during the history. Because of its high mortality rate, it must be diagnosed and treated at the earliest time post infection and therefore, rapid diagnostic tests are required. In the present study, we cloned the coding sequence of F1 capsular antigen of the bacteria in the pBAD/gIII plasmid for later expression and purification of the protein to produce poly and monoclonal antibodies against this antigen, and subsequently to develop rapid and efficient diagnostics tools for Y. pestis infections.

Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

Expression of interleukin-15 and inflammatory cytokines in skeletal muscles of STZ-induced diabetic rats: effect of resistance exercise training.

Skeletal muscle atrophy is associated with type-1 diabetes. Skeletal muscle is the source of pro- and anti-inflammatory cytokines that can mediate muscle hypertrophy and atrophy, while resistance exercise can modulate both muscle mass and muscle cytokine expression. This study determined the effects of a 5-week resistance exercise training regimen on the expression of muscle cytokines in healthy and streptozotocin-induced diabetic rats, with special emphasis on interleukin-15 (IL-15), a muscle-derived cytokine proposed to be involved in muscle hypertrophy or responses to stress. Induction of diabetes reduced muscle weight in both the fast flexor hallucis longus (FHL) and slow soleus muscles, while resistance training preserved FHL muscle weight in diabetic rats. IL-15 protein content was increased by training in both FHL and soleus muscles, as well as serum, in normal and diabetic rats. With regard to proinflammatory cytokines, muscle IL-6 levels were increased in diabetic rats, while training decreased muscle IL-6 levels in diabetic rats; training had no effect on FHL muscle IL-6 levels in healthy rats. Also, tumor necrosis factor-alpha (TNF-α) and IL-1ß levels were increased by diabetes, but not changed by training. In conclusion, we found that in diabetic rats, resistance training increased muscle and serum IL-15 levels, decreased muscle IL-6 levels, and preserved FHL muscle mass.

[Production and evaluation of immunologic characteristics of mzNLA-3, a non-infectious HIV-1 clone with a large deletion in the pol sequence].

Inactivation ofintegrase and reverse transcriptase can revoke the replication of HIV virions, and non-infectious HIV particles are desirable virus-like particle (VLP) vaccine candidates. Here, we produced inactive in replication HIV-1 particles fit for vaccine and virological purposes by introducing a mutation into the pol sequence. Proviral DNA (pNLA-3) was cut at two points in the pol region using the Bal I restriction enzyme and then religated. HEK 293T cells were transfected with the resultant plasmid (pmzNL4-3) to produce mutated virions. To confirm a production of VLPs and evaluate their biological activity the p24 load and syncytium formation (MT2 cells) were analyzed. The assay indicated that mzNL4-3 virions were assembled and contained functional envelope glycoproteins (ENV). In addition, mzNL4-3 virions were not able to infect MT2 and HEK 293T cells. Furthermore, the immunogenicity of VLPs was investigated in a mouse model. According to the data on vaccinated mice, the titer of ENV-specific antibodies rose rapidly after a boosting injection. Moreover, lymphoid cells extracted from these mice proliferated after exposure to the antigen. The mzNL4-3 virus particles possessed immunogenic antigens of HIV and can effectively trigger humoral and CD4 immune responses. Non-infectious mzNL4-3 virions may also be used in biomedical experiments to improve the biological safety conditions. Moreover, the mzNL4-3 seems to be a promising candidate for further HIV-1 vaccine investigations.

Optimization of a spray drying process to prepare dry powder microparticles containing plasmid nanocomplex.

Successful gene delivery to the lung depends not only on precise and effective design of a nanosized nucleic acid delivery system but also on well engineered liquid or solid microparticles. In present work, we tried to statistically optimize spray dried formulations of low molecular weight chitosan-plasmid nanocomplexes via a D-optimal design with respect to five critical responses: yield of the process, microparticle sizes, nanocomplex sizes, DNA stability and relative transfection efficiency. Nonocomplex formulations prepared by different amounts of solid contents and leucine ratio, and spray dried immediately with varying inlet temperature, feed rate and spray air flow rate. Mean results fitted to 2FI models except for relative transfection efficiency, which fitted in a quadratic model. According to the fitted models, the most important pure factors influencing each response determined to be feed rate for yield and DNA stability, feed fluid concentration for microparticle size, inlet temperature for nanoparticle size and leucine concentration for relative transfection efficiency. However, two-factor interactions have more important roles in microparticle size, nanocomplex size and DNA stability. It was concluded that the optimized formulation could be obtained when all the independent variables were at their maximum tested values, except for feed fluid concentration, which should be in its middle point.

Development of single-cycle replicable human immunodeficiency virus 1 mutants.

Non-infectious but antigenic human immunodeficiency virus 1 (HIV-1) particles are essential tool for the research on many topics associated with this virus. Here we report the construction of plasmid containing the HIV-1 genome mutated in the pol gene, which was co-transfected with plasmids expressing the pol gene products reverse transcriptase (RT) and integrase (IN), and the glycoprotein G of vesicular stomatitis virus (VSV-G). The virions produced in HEK 293 T cells were antigenic, but able to replicate only for one cycle, e.g. first generation single-cycle replicable (SCR) virions. The presence of VSV-G in the envelope of these virions had to ensure a wider spectrum of susceptible cell types for the replication of SCR. Replication of the first generation SCR virions in HEK 293T, MT-2, and mouse spleen cells was examined by p24-capture ELISA, syncytium formation assay, and electron microscopy (EM). HEK 293T and MT-2 cell lines showed a similar replication capacity, while primary cultures of mouse spleen cells were much less effective. The infection of MT-2 cells with the first generation of SCR virions yielded the second generation SCR virions, which were non-infectious. Summing up, the HIV-1 SCR virions represent the useful tool for HIV-1 research facilitating a better biological safety. Moreover, considering their antigenic composition and limited replication, SCR virions may be a promising candidate for the vaccine studies.

Biocompatibility evaluation of HDPE-UHMWPE reinforced ß-TCP nanocomposites using highly purified human osteoblast cells.

Biocompatibility of ß-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on ß-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on ß-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized ß-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering.

HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model.

UNLABELLED: Many Human immunodeficiency virus (HIV) candidate vaccines have been tested in clinical trials, but none was sufficiently effective in the prevention of HIV infection. A HIV vaccine should induce humoral as well as cell-mediated response, the latter including the cytotoxic CD8+ T lymphocyte (CTL) response. In this study, we immunized BALB/c mice with a purified fusion peptide Gag p24-Nef and evaluated immune responses. As for the cellular responses, the adjuvanted fusion peptide induced lymphocyte proliferation, CTL response, and cytokines IFN-gamma and IL-4 in the Th1 pattern. Humoral immune response to the adjuvanted fusion peptide included an increase in IgG antibodies of more IgG2a than IgG1 subtype. These results indicate that the employed HIV-1 peptide construct can elicit both cellular and humoral immune responses in mice. Further studies aimed at memory T cells and other aspects of immune responses are needed before a comprehensive assessment of this candidate vaccine could be provided. KEYWORDS: epitopes; fusion peptide; HIV-1 p24-Nef; immune response.

Multipotent mesenchymal stromal cells: optimization and comparison of five cationic polymer-based gene delivery methods.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are promising candidates in the field of regenerative medicine and in several studies have been genetically modified to bring a new property to or enhance an existing one in these cells. Furthermore, MSC have been used as gene delivery vehicles. The success of these experiments depends on selecting an appropriate method for gene delivery to the cells. METHODS: MSC were isolated from rat bone marrow; their authenticity was checked by differentiation experiments as well as staining for cell-surface markers. A systematic approach was used to optimize five cationic polymer-based gene delivery methods (Lipofectamin2000, Effecten, Superfect, Polyfect and FuGENE HD). The transfection yield and cell viability of each method was measured after 48 h in three to six separate experiments with nine to 12 different ratios and amounts of DNA/transfection reagent. RESULTS: The isolated MSC were successfully differentiated to osteoblasts, adipocytes and chondroblasts. They were positive for rat CD90 and CD73 and negative for CD31, CD45, CD11b and VEGFR2 markers. The average transfection rates with optimum conditions were 5.18+/-2.72 (FuGENE HD), 8.72+/-4.52 (Effecten), 9.59+/-3.12 (Superfect), 16.29+/-7.44 (Polyfect) and 19.60+/-3.12 (Lipofectamine 2000). The toxicity was below 20% for all reagents. DISCUSSION: Moderate levels of transfection and acceptable cell viability could be achieved using Lipofectamine 2000 and Polyfect in optimized conditions. The results could be improved by gating and sorting live cells using a simple FSC-SSC gating.

A novel single step double positive double negative selection strategy for beta-globin gene replacement.

beta-Thalassemias are a heterogeneous group of autosomal recessive disorders, characterized by reduced or absence of the beta-globin chain production by the affected alleles. Transplantation of genetically corrected autologous hematopoietic stem cell (HSC) is an attractive approach for treatment of these disorders. Gene targeting (homologous recombination) has many desirable features for gene therapy due to its ability to target the mutant genes and restore their normal expression. In the present study, a specific gene construct for beta-globin gene replacement was constructed consisting of: two homologous stems including, upstream and downstream regions of beta-globin gene, beta-globin gene lying between hygromycin and neomycin resistant genes as positive selection markers and thymidine kinase expression cassettes at both termini as negative selection marker. All segments were subcloned into pBGGT vector. The final plasmid was checked by sequencing and named as pFBGGT. Mammalian cell line COS-7 was transfected with linear plasmid by lipofection followed by positive and negative selection. DNA of the selected cells was analyzed by PCR and sequencing to confirm the occurrence of homologous recombination. In this novel strategy gene replacement was achieved in one step and by a single construct.