Photochromic Polyoxoniobates with Photoinduced "D-f-A" Electron Transfer Mechanism.

Compounds with redox activities have appealing applications in catalytic, electronic and magnetic properties, but the redox inert of polyoxoniobates (PONbs) significantly limits their applications for a long time. In this work, we are able to integrate organophosphate and lanthanide cluster into PONb to create the first family of inorganic-organic hybrid organophosphate-Ln-PONb composite clusters. These novel species not only present the first family of redox active PONbs that can be reduced to form long-lived "heteropoly blues" under ambient conditions, but also a new photochromic system. More importantly, the analyses of the electronic configurations and photochromic properties for a series of designed proof-of-concept PONbs models allow us to discover a D-f-A electron transfer mechanism, that is, photoinduced electron is transferred from a photosensitive organophosphate electron donor (D) to the NbV electron acceptor (A) through the unoccupied 4 f-orbitals of Ln (f). This work paves the way for developing diverse PONb-based redox materials and expanding the possibility of the applications of PONbs in the redox chemistry.

LncRNA SNHG5 promotes the glycolysis and proliferation of breast cancer cell through regulating BACH1 via targeting miR-299.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors in women. Accumulating studies have been reported that long non-coding RNA (lncRNA) SNHG5 is highly expressed in BC. However, the specific molecular mechanism of SNHG5 in BC is unclear. METHODS: Gene and protein expressions in BC cell were detected by qRT-PCR and western blotting. The proliferation and cell cycle were measured using colony formation assay and flow cytometry analysis, separately. The glucose consumption and lactate production were determined by using the glucose assay kit and lactate assay kit. A dual-luciferase reporter assay was performed to measure the interaction between miR-299 and SNHG5 or BACH1. RESULTS: SNHG5 and BACH1 expressions were increased in BC cell while miR-299 level was decreased. SNHG5 increased BACH1 expression by directly targeting miR-299. SNHG5 silencing or miR-299 overexpression suppressed the proliferation of BC cell, arrested the cell cycle in the G1 cell phase, and decreased the glucose consumption and lactate production of BC cell. However, inhibition of miR-299 or overexpression of BACH1 could reverse the inhibitory effects of sh-SNHG5 on cell proliferation and glycolysis in BC. CONCLUSION: SNHG5 promoted the BC cell growth and glycolysis through up-regulating BACH1 expression via targeting miR-299. These findings may improve the diagnostic and therapeutic approaches to BC.

Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35.

Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy.

[Extraction of miRNA from Glycyrrhiza uralensis Decoction and Its Effect on Immune Cells].

OBJECTIVE: To extract microRNA(miRNA) from Glycyrrhiza uralensis(liquorice) decoction and to explore its effect on mmune cells. METHODS: With dried processed liquorice, the water decoction was prepared according to the conventional method and subsequently concentrated by rotary evaporation. The concentrated decoction was further freeze-dried by freeze dryer, and miRNAs were extracted with Plant MicroRNA Extraction Kit. The extracted miRNA was digested by DNase I and then analyzed through the agarose gell electrophoresis. The PBMC was isolated from healthy volunteers and treated respectively by liquorice water extract, glycyrrhizic acid, glycyrrhetic acid and liquorice miRNAs. After 24 hours, the cells numbers were counted, and the changes of cell morphology were observed. The expression of CD3, CD56 and HLA-DR were analyzed by flow cytometry to identify the change of cell subsets in PBMC. RESULTS: miRNAs could be extracted from the decoction of dried liquorice which further confirmed the stability of miRNAs. The in vitro culture experiment showed that,compared with the controls, PBMC treated with liquorice miRNAs appeared apparent cell aggregation and increased cell number and HLA-DR+ cells proportion. CONCLUSION: The miRNAs are successfully extracted from the freeze-dried decoction of dried liquorice. It is indicated that liquorice miRNAs have significant stimulative effects on the growth of PBMC and HLA-DR+ cells subset.

[Experimental study on anti-pancreatic cancer effect of oridonin].

OBJECTIVE: To investigate the apoptotic effect of oridonin in human pancreatic cancer cells PANC-1, and to explore the underlying mechanism. METHODS: MTT assay was used to measure the cell viability. Apoptosis was determined by confocal laser scanning microscope after Hoechst 33342 staining and flow cytometry analysis after PI staining. The regulation of JNK and p38 MAPK signaling pathway proteins was examined by Western blot analysis. RESULTS: Treatment with oridonin for 24 h resulted in a marked decrease in cell viability in a dose-dependent manner. The IC50 value was determined as 49.80 µmol/L for 24 h. After treatment with 50 micromol/L and 80 µmol/L oridonin for 24 h, typical apoptotic nucleus alterations were observed with confocal laser scanning microscope and apoptotic rates of PANC-1 cells increased by flow cytometry analysis. Treatment with 80 µmol/L oridonin down-regulated protein expression of JNK, p38 and increased the expression of p-JNK, p-p38. Furthermore, 80 µmol/L oridonin treatment decreased the expression of down-stream proteins Caspase-9, Caspase-3 and PARP in the apoptotic pathway as well as activated the cleavage of Caspase-9. CONCLUSION: Oridonin can induce apoptosis of PANC-1 cells through JNK and p38 MAPK pathway proteins.

Chimeric adenoviral vector Ad5F35L containing the Ad5 natural long-shaft exhibits efficient gene transfer into human T lymphocytes.

Adoptive therapy using T cells modified with tumour antigen-specific T cell receptor (TCR) genes has become a popular area of research in tumour biotherapy research. However, the efficiency of this treatment is low. To increase the efficiency of this therapy, the antigen specific TCR expression in the T cells needs to be improved. Adenoviral vector-mediated gene expression is an attractive approach to bypass the issue of TCR gene modification. The efficiency of adenovirus vector serotype 5 (Ad5) infection is low due to the absence of coxsackievirus B-adenovirus receptor (CAR) expression in T cells. In the present study, a chimeric adenoviral vector (Ad5F35L) was generated; this construct contained both the natural long-shaft of Ad5 and the Ad35 knob. A transduction study showed that the Ad5F35L vector exhibited a higher transduction efficiency in human primary T lymphocytes than the Ad5 vector and the Ad5F35S vector, which contained the Ad35 natural short-shaft and the Ad35 knob. Similar transduction efficiencies were observed for both CD4(+) T lymphocytes and CD8(+) T lymphocytes and the transfection was independent of the expression of cell surface receptors. The activation of T lymphocytes resulted in an improvement of the Ad5F35L transduction efficiency in CD4(+) T cells and a decrease in Ad5F35L transduction efficiency in CD8(+) T cells. The results demonstrate that Ad5F35L is a promising viral vector and will facilitate the clinical application of tumour antigen-specific TCR gene therapy.

Forced expression of indoleamine-2,3-dioxygenase in human umbilical cord-derived mesenchymal stem cells abolishes their anti-apoptotic effect on leukemia cell lines in vitro.

The ability of mesenchymal stem cells (MSCs) to preserve cancer cells potentially constitutes the adverse effect of MSC-based cell therapy in the context of hematologic malignancy. In an effort to reverse this undesirable feature of MSCs, we manipulated human umbilical cord-derived MSCs (UC-MSCs) to express indoleamine-2,3-dioxygenase (IDO), an enzyme that induces immune suppression by inhibiting T cell proliferation and triggering apoptosis in immune cells. Cultures of human UC-MSCs were generated by plastic adherence method. Full-length cDNA of human IDO was cloned into adenovirus shuttle vector. Then, the recombinant virus harboring IDO gene was produced in 293 cells and used to infect UC-MSCs. Expression of IDO protein was detected within infected UC-MSCs, and accumulation of kynurenine was observed in the supernatant. Two human leukemia cell lines, Jurkat and HL-60, were cultured on the monolayer of native or infected UC-MSCs, respectively. It was observed that forced IDO expression abolished the anti-apoptotic effect of UC-MSCs on these leukemia cells and enhanced their proliferation inhibitory effect on activated human lymphocytes as well as leukemia cells. These results suggested that equipping MSCs with IDO could be one of the reasonable strategies to reverse their cancer-supportive effect unfavorable for clinical applications.

Identification of a novel HLA-A2-restricted mutated Survivin epitope and induction of specific anti-HCC CTLs that could effectively cross-recognize wild-type Survivin antigen.

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.

Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates.

[Gas chromatography-mass spectrometric analysis of Jatropha curcas leaf extracts prepared by supercritical fluid CO2 extraction].

OBJECTIVE: To analyze the bioactive components in Jatropha curcas leaves using gas chromatography-mass spectrometry (GC-MS). METHODS: The bioactive components were extracted from J. curcas leaves by supercritical fluid CO2 extraction and analyzed by using GC-MS. RESULTS: Seventy peaks were detected by GC-MS, and 43 compounds were identified (61.43%). Among the identified compounds, 16 had a content of more than 1%, and the total contents of these 16 compounds reached 81.36%. The four most abundant components were 22,23-dihydro-stigmasterol (16.14%), alpha-tocopherol (15.18%), beta-amylin (7.73%) and dotriacontanol (7.02%). The content of gamma-tocopherol reached 2.88% and vitamin E reached 18.06% in the extract. CONCLUSION: J. curcas leaves contain multiple compounds with anti-tumor, anti-virus and antimicrobial activities.

[Tumor-specific T cell recptor gene transfection promotes memory T cell differentiation in vitro].

OBJECTIVE: To investigate effect of tumor-specific T cell receptor gene transfection on memory T cell differentiation in vitro. METHODS: TCRVbeta7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vbeta7.1 was detected by flow cytometry before and after the transfection. Memory T cell differentiation was induced by stimulation with the hepatocarcinoma cell line BEL-7402 in vitro. The expression of surface molecules CD45RO, CD45RA and CCR7 was analyzed by flow cytometry to identify the phenotype and subsets of the memory T cells. Fluorescence-activated cell sorting was performed to detect the apoptosis of the tumor cells, and enzyme-linked immunoabsorbent assay was used to determine the production of interferon-gamma (IFN-gamma) for assessing the immune function of the memory T cells. RESULTS: Flow cytometry showed that TCRVbeta7.1 gene was efficiently expressed after transfection. After stimulation by the tumor cells in vitro, the expression of CD45RO in TCRVbeta7.1 gene-modified T cells increased gradually, and analysis of the coexpression of CD45RA and CCR7 revealed that the effector memory T cells constituted the majority of the differentiated memory T cells. The apoptotic rate of the tumor cells induced by the T cells increased significantly with also obviously increased INF-gamma secretion in the memory T cells. CONCLUSION: Tumor-specific TCRVbeta7.1 gene transfection can promote the differentiation of the memory T cells, the majority of which belongs to effector memory T cells that perform immune functions by inducing apoptosis and cytokine secretion.