Production of recombinant Human T Lymphotropic Virus type 1 Tax protein in Rosetta (DE3) bacterial host

 HTLV1 is the first detected retrovirus causing disease in human. The physiopathology of HTLV1 related diseases was mainly linked with its Tax protein characteristics. Use of mutant Tax proteins accompanied by immune regulator drugs could help treating HTLV1 associated myelopathy patients as a modulator of potent immune response against this viral protein. Since Tax protein is not commercially available, production of recombinant Tax protein was targeted for this study. Coding sequence of Tax protein (containing R222K mutation) in the pcDNA3.1(+) was digested with BamHI and XhoI restriction enzymes, and then removed and inserted into the expression vector pET32a(+) within the same cutting sites and cloned into E.coli DH5α. Recombinant vector was confirmed with enzymatic digestion, colony PCR, and sequencing of cloned gene. E.coli Rosetta (DE3) was transformed with the recombinant plasmid and the expression was induced. The expression of protein was assayed with SDS-PAGE and western blot using monoclonal antibodies against Tax and 5His epitope. Finally, antigenic characteristic of the recombinant protein was evaluated by western blotting against patient sera. Presence of Tax protein band in the SDS-PAGE and western blot was confirmed. Western blotting of the recombinant protein with patient sera showed the band related to Tax protein. The recombinant protein is well produced and could be detected by patients' sera, making it eligible to be used as a recombinant viral antigen for future purposes

Gene Delivery to Mesenchymal Stem Cells

There is increasing trend in using recombinant stem cells as novel therapeutic candidates in different diseases. These studies encompass different applications from targeted homing of Mesenchymal Stromal (stem) Cells (MSC), to arming them with different cytokines. Resistance to transfection or transduction methods had urged researchers to look for better gene delivery alternates and optimizing them. Though chemical transfection methods are usually considered safer than viral gene delivery methods, most of these reagents suffer from low efficiency in lower concentrations and high toxicity in the higher ones. We, as well as other researchers, have reported the best efficiencies with Lipofectamin 2000TM reagent, with up to 50% efficiency in some reports while we have not been able to reach this level. Theoretically low transfection efficiency could be compensated by stably transfecting a cell line followed by long term culture in a selective medium. Usually this approach is not practical for MSC since they should be used within the first few passages after isolation. Indeed, we have previously shown that long-term culture of these cells is associated with chromosomal abnormalities and profound morphological changes.  Lentiviral transduction methods have achieved the highest efficiency in delivering foreign genes to MSC ( above 95%in several cases) but the safety concerns has hindered their application in clinical studies. While adeno-associated viral vectors have been used in several gene therapy studies, MSC seem to be resistant to this method. There are reports of high efficiency of adenoviral gene delivery to MSC, though in our hand it was much lower than lentiviruses. Howevere, we found pretreating these cells with valproic acid could increase transduction efficiency of adeno-associated vectors by 2.5 fold, mainly through increasing the expression of its cell surface receptor. In our lab, using lentiviral vectors, we could transduce MSC with the efficiency of more than 90%.  In conclusion, we believe that chemical transfection methods such as Lipofectamin 2000TM is a good choice when transduction rate is not a main concern and lentiviral vectors are suitable for a high yield stable gene delivery to these stem cells. Keywords: Mesecnchyal Stromal Cells, Gene Transfere Techniques

Ethanolic extract of <i>Ferula gummosa</i> is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest

<div><p><i>Ferula gummosa</i> Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of <i>F. gummosa</i> as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC₅₀ value at 72 h (0.001 ± 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ± 0.037, 0.01 ± 2.51 mg/g of dry powder). Generally, the results obtained indicate that <i>F. gummosa</i> ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent.</p></div