Traditional T vector cloning method requires onerous procedures for identifying recombinant, and directional cloning was impossible. In order to overcome these problems, we have devised a directional T vector pETG based on pET-23a(+). For gene cloning, 7 bp partial LacO sequence was introduced into DNA fragment to reconstitute a full length LacO with Bfu I digested T vector. After transformation, blue colonies were selected on LB plate supplemented with X-gal. Restriction enzyme digestion and PCR identification showed that all blue colonies contained the directionally inserted recombinants and the recombinant efficiency was nearly 100%. We have successfully cloned 103 genes from human liver cDNA; in the study complicated procedures for screening of recombinant were not required. Eight pETG clones were picked for protein expression, and all the clones successfully produced corresponding proteins. We demonstrated that the directional T vector was successfully constructed, and it was very suitable for gene cloning and expression.
Cloning, Molecular ; methods ; DNA, Complementary ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; genetics ; Genetic Vectors ; genetics ; Humans ; Liver ; chemistry ; Polymerase Chain Reaction ; methods ; Recombinant Proteins ; biosynthesis ; genetics

In order to investigate the effects of pyrolysis conditions on bio-oil production from biomass in molten salt, experiments of biomass pyrolysis were carried out in a self-designed reactor in which the molten salt ZnCl2-KCl (with mole ratio 7/6) was selected as heat carrier, catalyst and dispersion agent. The effects of metal salt added into ZnCl2-KCl and biomass material on biomass pyrolysis were discussed, and the main compositions of bio-oil were determined by GC-MS. Metal salt added into molten salt could affect pyrolysis production yields remarkably. Lanthanon salt could enhance bio-oil yield and decrease water content in bio-oil, when mole fraction of 5.0% LaCl3 was added, bio-oil yield could reach up to 32.0%, and water content of bio-oil could reduce to 61.5%. The bio-oil and char yields were higher when rice straw was pyrolysed, while gas yield was higher when rice husk was used. Metal salts showed great selectivity on compositions of bio-oil. LiCl and FeCl2 promoted biomass to pyrolyse into smaller molecular weight compounds. CrCl3, CaCl2 and LaCl3 could restrain second pyrolysis of bio-oil. The research provided a scientific reference for production of bio-oil from biomass pyrolysis in molten salt.
Biofuels ; analysis ; Bioreactors ; microbiology ; Catalysis ; Chlorides ; chemistry ; Lanthanum ; chemistry ; Lipids ; biosynthesis ; Oryza ; metabolism ; Plant Stems ; metabolism ; Potassium Chloride ; chemistry ; Salts ; chemistry ; Zinc Compounds ; chemistry

Objective:To investigate the expression of long non-coding RNA(lncRNA) ZFP36-AS1 in bladder cancer and the effect of ZFP36-AS1/miR-221 axis on the proliferation and immune escape of bladder cancer cells.Methods:The expression difference of ZFP36-AS1 in bladder cancer tissues was analyzed by cBioPortal database. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression difference of ZFP36-AS1 in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637). MGH-U3 cells were randomly divided into negative control (NC) group and ZFP36-AS1 group, which were transfected with pcDNA3.1-NC plasmid and pcDNA3.1-ZFP36-AS1 plasmid, respectively. Colony formation assay and flow cytometry were used to analyze the proliferation activity and cell cycle of MGH-U3 cells, respectively. T lymphocytes were co-cultured with MGH-U3 cells in each group, and the levels of interleukin-10 (IL-10), γ-interferon (IFN-γ), and interleukin-4 (IL-4) in the supernatants of each group were detected by enzyme-linked immunosorbent assay (ELISA). The dual-luciferase reporter gene assay verified the targeting relationship between ZFP36-AS1 and miR-221. The effect of ZFP36-AS1 on the expression of miR-221 in MGH-U3 cells was detected by RT-qPCR. Western blotting was used to detect the effect of ZFP36-AS1/miR-221 axis on the protein expression of CDK3, Cyclin C, CDK5, Cyclin D1 and Cyclin D3 in MGH-U3 cells.Results:Compared with normal bladder tissue, ZFP36-AS1 was abnormally low-expressed in bladder cancer tissue ( P<0.01). Compared with SV-HUC-1 cells, ZFP36-AS1 was abnormally low-expressed in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637) ( P<0.01), and the expression was lowest in MGH-U3 cells ( P<0.01). The number of MGH-U3 cell colonies formed in the NC group and the ZFP36-AS1 group were (220.80±34.65) and (77.84±19.11), respectively, and the number of MGH-U3 cell colonies formed in the ZFP36-AS1 group was significantly down-regulated, the difference was statistically significant ( P<0.01). The proportions of G 0/G 1 phase cells in NC group and ZFP36-AS1 group were (48.04±2.89)% and (72.89±3.46)%, respectively, and the proportion of S phase cells were (35.38±2.98)% and (20.62±2.56)%, respectively. The proportion of G 2/M stage cells was (16.59±1.46)% and (6.48±1.50)%, respectively. The proportion of cells in G 0/G 1 phase were up-regulated in ZFP36-AS1 group ( P<0.01), and the proportion of cells in S phase and G 2/M phase were both down-regulated ( P<0.01). Compared with the NC group, the levels of IL-4 and IFN-γ in the ZFP36-AS1 group were significantly up-regulated ( P<0.01), and the level of IL-10 was significantly down-regulated ( P<0.01). ZFP36-AS1 can target miR-221 ( P<0.01). The relative expression of miR-221 in the NC group and the ZFP36-AS1 group was 6.84±1.35 and 1.00±0.21, respectively. Compared with the NC group, overexpression of ZFP36-AS1 could significantly inhibit the expression of miR-221 ( P<0.01). Compared with the NC group, the expressions of CDK3, Cyclin C, CDK5, Cyclin D1, and Cyclin D3 in the ZFP36-AS1 group were significantly decreased. Conclusion:ZFP36-AS1 is abnormally low-expressed in bladder cancer, and it reduces the proliferation activity of bladder cancer cells and inhibits their immune escape by inhibiting the expression of miR-221.

Objective: To investigate the effects of milrinone on levels of inflammatory factors and liver and renal function after CPB in rheumatic heart disease patients for valve replacement. Methods: From January 2014 to January 2016, 80 patients received valve replacement in the Central Hospital of Chongqing Three Gorges were randomly divided into observation group and control group by block randomization grouping method, with 40 patients in each group.The patients in the observation group were pumped intravenously with milrinone 0.5μg·kg^-1·min^-1 for 72h after surgery, while the patients in the control group were not pumped.The serum levels of IL-6, IL-8, IL-10, TNF-α were detected by ELISA before operation and on 0d, 1d, 3d, 5d after operation, respectively.The levels of ALT, AST, Scr were also detected at the same time.Moreover, the time for operation, extracorporeal circulation, interruption, mechanical ventilation, ICU and hospital were also compared between the two groups. Results: The levels of TNF-α, IL-6, IL-8 and IL-10 increased immediately after operation in both groups[control group: (14.97±5.14)pg/mL, (52.45±10.37)pg/mL, (34.10±8.38)pg/mL, (32.27±8.45)pg/mL; observation group: (16.05±5.71)pg/mL, (54.39±8.56)pg/mL, (33.80±7.69)pg/mL, (31.48±5.94)pg/mL, t=-0.628, -0.644, 0.116, 0.342], and the peak values of TNF-α, IL-6 and IL-8 reached on the first day after operation in both two groups[control group: (52.07±10.18)pg/mL, (96.04±26.45)pg/mL, (91.14±18.28)pg/mL, (48.10±9.78)pg/mL; observation group: (50.37±12.98)pg/mL, (93.66±24.10)pg/mL, (83.16±16.28)pg/mL, (46.68±9.25)pg/mL, t=0.559, 0.295, 1.458, 0.473], and the peak value of IL-10 reached on the 3rd day after operation(t=-3.577), the differences were statistically significant on the 3rd day after operation[control group: (36.03±9.39)pg/mL, (59.56±14.38)pg/mL, (53.91±13.16)pg/mL, (85.55±16.49)pg/mL; observation group: (36.70±4.33)pg/mL, (36.20±3.85)pg/mL, (42.91±7.30)pg/mL, (101.33±10.81)pg/mL, t=-0.289, 7.017, 3.267, -3.577]. The levels of ALT, AST and Scr increased immediately after operation in both groups[control group: (38.51±5.12)U/L, (40.23±5.03)U/L, (62.27±5.02)μmol/L; observation group: (39.20±4.67)U/L, (39.6±4.94)U/L, (73.61±4.04)μmol/L, t=0.114, 0.243, 0.630], there were tatistically significant difference between the two groups on the 5th day after operation[control group: (61.45±5.27)U/L, (54.20±7.0)U/L, (86.45±9.01)μmol/L; observation group: (36.20±3.85)U/L, (34.85±7.12)U/L, (83.7±11.07)μmol/L, t=11.231, 9.224, 5.647], and on the fifth day, the levels of ALT, AST and Scr in the observation group dropped to normal, while only the level of Scr in the control group dropped to normal.There were no statistically significant differences in the time of operation, extracorporeal circulation, interruption, mechanical ventilation between two groups (t=0.267, 0.151, 0.187, 0.773, all P>0.05). However, there were statistically significant differences in the time of ICU and hospital [control group: (54.90±16.84)h, (14.35±3.01)d, observation group: (44.05±7.06)h, (10±1.86)d, t=8.149, 13.042, all P<0.05]. Conclusion Milrinone can obviously improve the inflammatory reaction in surgical trauma tissues caused by IL-6, IL-8, IL-10, TNF-α, and can protect liver, renal tissue from injury, moreover, it can decrease the incidence of postoperative complications and the length of hospital stay.