Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

A simple and highly efficient method for transduction of human adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a wide range of cell types and provide a potential to transfer therapeutic protein in vivo, making them valuable candidates for gene therapy and cell therapy. However, using MSCs in in vivo is limited due to the low rate of transfection and transduction efficacy. Therefore, developing methods to efficiently transfer genes into MSCs would provide a number of opportunities for using them in the clinic. Here, we introduce a simple and robust method for efficient transduction of human adipose-derived MSCs by modification under the culture condition of human embryonic kidney cells 293 (HEK293T) and MSCs. Moreover, as a transduction enhancer, polybrene was replaced with Lipofectamine, a cationic lipid. Therefore, we showed that transduction of primary cells can be increased efficiently by modifying the culture condition.     

c-src在大鼠卵巢中的表达及其在原始卵泡生长启动中的作用初探

目的：研究原癌基因c-src在大鼠卵巢的表达,及其在原始卵泡启动过程中的作用。方法：取2日龄SD雌性大鼠卵巢,在Waymouth培养体系中培养0．4、8d后,首先采用RT-PCR方法证实大鼠卵巢中有c-src的表达,再体外合成其RNA小干扰片段（small interference RNA,siRNA）转染培养中的卵巢组织进行RNA干扰,用HE染色及RT-PCR筛选最佳干扰片断并用慢病毒包装后检测干扰效果。结果：随着培养天数的增加,原始卵泡在卵泡总数中所占比例逐渐减少;c—src mRNA在原始卵泡中有表达,经筛选用最佳干扰片断siRNA1慢病毒包装进行RNA干扰,发现干扰后,与空白组、空白载体组相比,最佳干扰组c—src mRNA含量明显下降,原始卵泡在卵泡总数中所占比例相对更多,原始卵泡发育受到抑制。结论：c-src在原始卵泡中有表达,并在一定程度上促进了原始卵泡的发育。     

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10¹¹ bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by ‘scrape loading’. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.     

Silencing of Inducible Immunoproteasome Subunit Expression by Chemically Modified siRNA and shRNA

Overexpression of inducible subunits of immunoproteasome is related to pathogenesis of some chronic diseases. Specific inhibition of the immunosubunits may be used for the treatment of these diseases and RNA interference is one of the potent methods used in this area. We designed 2′-O-methyl modified siRNAs with selectively protected nuclease-sensitive sites, which efficiently silence LMP2, LMP7, and MECL-1 genes expression. To provide stable long-lasting inhibition of target genes, short-hairpin RNAs (shRNA) expressed by lentiviral vectors were constructed. Our results demonstrated that chemically modified siRNAs inhibited the expression of target genes with similar efficiency or with efficiency exceeding that of corresponding shRNAs and provide silencing effect for 5 days.     

Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.     

Molecular characterization and comparison of simian immunodeficiency virus isolates from macaques, mangabeys, and African green monkeys

Simian immunodeficiency virus (SIV)/Mne has been inoculated into three species of macaques and into baboons. Virus was isolated from all the macaques who subsequently died at 15 to 120 weeks (mean 80 weeks) with various manifestations of immune deficiency. Individual animals varied in their viral antibody profile as a function of time after infection. Independent SIV isolates obtained from African green monkeys and magabeys were compared to SIV/Mne for their ability to replicate in lymphocytes and macrophages and with respect to the immunological relatedness of their viral proteins. Antibodies present in human immunodeficiency virus-2 (HIV-2)-infected individuals were readily detected by the virus produced by a single-cell clone of SIV/Mne.