Objective ToexpresschimericantigenofHCVwithmultipleimmunodominantepitopes inE .coliforimprovingthequalityofreagentsforHCVscreening .Methods Thegenefragmentencoding HCVchimericantigenwasobtainedbymolecularcloningmethodandclonedintopQE 30 plasmidforex pressioninE .coliM 15 .TheexpressedchimericantigenwaspurifiedbyNi NTAandcoatedonELISA platestoanalyzeitssensitivityandspecificity .Results Thechimericantigenof 5 30 0 0washighlyex pressed .ELISAassayofserumsamples (including 18HCVpositiveand 17negativesera)indicatedthatthe HCVchimericantigenhadhighsensitivityandspecificity .Conclusions ChimericantigenofHCVwith typicalimmunodominantepitopescanbeusedtodevelopgoodreagentsforHCVimmunoassay .

Objective To explore the relationship between mitochondrial DNA deletion and malignant phenotypes of human lung cancer cells. Methods Two rho? derivatives of 95C and 95D were generated by treating the cultured cells with ethidium bromide. Agarose colony formation assays and Transwell invasion assays were carried out to detect the phenotypes of colony formation and invasiveness of the cultured cells, respectively. Cell growth was determined by MTT. Results The partially mtDNA-deleted cells exhibited stronger capacity of colony formation and invasiveness, and faster growth rates than their respective parental cell lines. Conclusion Mitochondrial DNA deletion might play a role in the formation of malignant phenotypes of human lung cancer.

Objective Curcumin and MS-275,an inhibitor of histone deacetylase (HDAC),are promising anti-tumor agents. The aim of present study was to investigate the mechanisms of apoptosis induced by combined use of MS-275 in low dosage and curcumin in prostate cancer cell line DU145. Methods MTT assay was used to evaluate the lethal effect on DU145 cells by solitary use of MS-275 and or combined with curcumin. The changes in cell life cycle was detected by flow cytometry. The expressions of the survival signaling pathways were determined by Western blotting. Results Solitary application of MS-275 or curcumin may inhibit the growth of DU145 cells in a time and dose-dependent manner. The combination of MS-275 (1?mol/L) and curcumin (20?mol/L) exhibited obvious cytotoxic effect on cell viability,which was only 45.9% 48h after the combined treatment. Cell cycle assay showed that the combination of MS-275 and curcumin resulted in obvious appearance of sub-G1 phase in DU145 cells,implying that the cell apoptosis had been induced. The results of Western blotting showed that after treatment of MS-275 or curcumin singly,the phosphorylation level of Akt and ERK kinase declined slightly,however,when MS-275 and curcumin were used together,there appeared a prominent inhibitory effect on Akt and ERK kinase,indicated by a sharp decline of their phosphorylation level,and at the same time,the level of cleaved PARP,a hallmark of apoptosis,was increased in DU145 cells. Conclusion Combined use of MS-275 and curcumin may exert a synergistic cytotoxic effect on the viability of DU145 cells,and exhibit an inhibitory activity on Akt and ERK kinases to induce apoptosis.

Protein tyrosine phosphatase (PTP) 1B is a potential target for the treatment of diabetes and obesity. Phosphotyrosine (pTyr) is the substrate for PTP1B dephosphorylation. Malonic acid moiety was used herein as a mimic of the phosphate group in pTyr, and novel malonic acid derivatives 1-7 were designed, synthesized and evaluated as PTP1B inhibitors. Results from enzymatic assays indicated that compounds 3 and 4 exhibited potent inhibition against human recombinant PTP1B with IC50 values of 7.66 and 1.88 micromol x L(-1), respectively.

Objective:To investigate the presence of SENV infection among patients in China,and analyze partial nucleotide sequence of SENV isolated from a patient with non A-G hepatitis.Methods:A nested polymerase chain reaction (PCR) assay with primers from ORF1 of SENV genome was established to detect SENV DNA.The PCR product was cloned and sequenced.Results:SENV DNA was positive in 2 of 7 patients with non A-G hepatitis and TTV negative.Partial gene of a SENV isolate was compared with the corresponding region of SENV isolate(AX025730)from Italy and was found that the nucleotide homology was 90%.Conclusions:The results of this study confirmed the presence of SENV infection in China.The development of a PCR assay for SENV DNA detection and the cloning,sequencing of the SENV isolate have important implication for the diagnosis and epidemiological investigation on SENV infection.

Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .

Objective To identify the role of phosphatidylinositol-3-kinase(PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) and the involved mechanism.Methods Knockdown of p110α,receptor-interacting protein 1(RIP1) or both p110αand RIP1 was mediated by the specific short hairpin RNA (shRNA) lentivirus and verified by RT-PCR or Western blotting .In addition , Western blotting was used to detect phosphorylation of mixed lineage kinase domain-like protein(MLKL) and protein kinase B(AKT) or tetramerization of MLKL.Cell death was measured by micros-copy and flow cytometry.Results AKT phosphorylation and TNFα-induced necroptosis of L929 cells were suppressed by the inhibitors of PI3K or AKT, as well as p110αknockdown.Moreover, RIP1 knockdown did not inhibit L929 cell death induced by TNFαplus Z-VAD, but the RIP1-independent necroptosis was inhibited by p 110αknockdown.In addition, p110αknockdown suppressed MLKL phosphorylation and tetramerization induced by TNFαwith Z-VAD in L929 cells. Conclusion PI3K mediates necroptosis of L929 cells induced by TNFαby activating AKT and MLKL, respectively.

BACKGROUNDTo isolate and identify the genes related to cancer metastasis by comparison of two cell strains with different metastasis potentials subcloned from human lung giant cell carcinoma cell line.

METHODSSuppression subtractive hybridization (SSH) was used to compare the levels of gene expression between the two cell strains and SSH library was constructed. After screening the library by gene chip, the expressed sequence tags (ESTs) with different expressing level were sequenced and blasted with GenBank.

RESULTSSeventy-nine genes were obtained that were expressed much higher in PLA-801D than in PLA-801C, including two full-length cDNA. GenBank Accession numbers of the two cDNA, named MAG-1 and MAG-2, were BC006236 and BC002420, the 8.5 kb MAG-1 gene was composed of four exons and located on the chromosome of 4q21. The MAG-2 gene, which was made up by 9 exons, had a length of 5.2 kb and its location was 2q35. Both sequences had open reading frames (ORF) and promoters before the theoretical transcription start points. Using special software, the secondary structure of theoretical products of the two cDNAs was prognosticated, α-helix was the main proportion, but β-pleated sheet and random coil were also included.

CONCLUSIONSThe expression of MAG-1 and MAG-2 has significant differences in these two cell strains, so they might impact tumor metastasis in some ways that are still uncharted.

OBJECTIVETo investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.

METHODSThe DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.

RESULTSThe vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.

CONCLUSIONElectroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Animals ; Antibody Formation ; Antibody Specificity ; Antigens, Neoplasm ; genetics ; immunology ; Electroporation ; Gene Fusion ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; HEK293 Cells ; Humans ; Injections, Intramuscular ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; genetics ; immunology

Objective:To explore the prognostic factors associated with clear cell adenocarcinoma (CCAC) of the uterine cervix based on data in the Surveillance, Epidemiology and End Results (SEER) database.Methods:Clinical data were collected from 431 patients with confirmed CCAC in the SEER database from 1976 to 2017. Survival analysis was performed using the Kaplan-Meier method with log-rank test for comparison between subgroups. Cox proportional hazards model was used to analyze the influencing factors of overall survival (OS).Results:The median age [ M ( Q1, Q3)] of 431 patients was 54 years old (40 years old, 71 years old); there were 333 cases (77.3%) of whit. The median OS time of 431 patients was 93 months (95% CI: 47-148 months), and the 1-, 2-, and 5-year OS rates were 80.1%, 65.8% and 54.2%, respectively. The median OS time was not reached in patients with American Joint Committee on Cancer (AJCC) stage Ⅰ, 83 months (95% CI: 21-144 months) for stage Ⅱ, 32 months (95% CI: 16-47 months) for stage Ⅲ, and 9 months (95% CI: 5-13 months) for stage Ⅳ ( P < 0.001). Median OS time was not reached in patients with SEER stage of localized lesions, 46 months (95% CI: 8-83 months) for regional lesions stage, and 9 months (95% CI: 5-12 months) for distant metastases stage ( P < 0.001). Of the patients with clear AJCC staging and some with unspecified AJCC staging, 118 received surgical treatment alone and 119 received postoperative radiotherapy, the median OS time of the two groups was 443 months (95% CI: 162-723 months) and 102 months (95% CI: 75-129 months), and the difference in OS between the two groups was statistically significant ( P < 0.001). Among the patients with AJCC stage Ⅰ, the 5-year OS rates in surgery-only group and postoperative radiotherapy group were 82.5% and 78.5%, the stage Ⅱ were 80.0% and 52.3%, and the stage Ⅲ were 27.8% and 63.3%, respectively; the differences in OS between different stages were not statistically significant (all P>0.05). Among the patients with SEER localized lesions stage, the 5-year OS rates in surgery-only group and postoperative radiotherapy group were 88.9% and 73.1%, and the difference was statistically significant ( P = 0.012); the regional lesions stage were 45.5% and 60.0%, and the difference was not statistically significant ( P = 0.568). The results of multivariate Cox regression analysis showed that AJCC staging (stage Ⅰ vs. stage Ⅳ, HR = 0.281, 95% CI: 0.178-0.543, P < 0.001; stage Ⅱ vs. stage Ⅳ, HR = 0.347, 95% CI: 0.113-0.439, P < 0.001; stage Ⅲ vs. stage Ⅳ, HR = 0.399, 95% CI: 0.030-0.145, P < 0.001), SEER staging (localized lesions stage vs. distant metastases stage, HR = 0.104, 95% CI: 0.059-0.182, P < 0.001; regional lesions stage vs. distant metastases stage, HR = 0.301, 95% CI: 0.195-0.463, P < 0.001) and whether or not receive surgery (yes vs. no, HR = 0.359, 95% CI: 0.241-0.535, P < 0.001) were independent influencing factors of OS in CCAC patients. Conclusions:AJCC staging, SEER staging and surgery are independent influence factors for OS in patients with CCAC, and postoperative radiotherapy may not provide more survival benefit.