Objective To clone the aldehyde dehydrogenase (adhA) gene from Methylovorus glucosotrophus and study its expression,purification and enzymatic characteristics.Methods The adhA gene was amplified and cloned to the expression vector pTIG.The AdhA was successfully expressed with induction in Escherichia coli BL21(DE3).The enzymatic characteristics were investigated by AHMT,and AdhA was purified by Ni+ exchange chromatography.Results AdhA accounted for more than 50% of the total cell proteins,and the purity was about 95%.With methanol as the substrate,the optimal pH of AdhA was 7.0,while the optimal temperature was 30℃.The enzymatic activity of purified AdhA remained about 60% when stored at room temperature for 6 days.Conclusion AdhA from MP688 is expressed in vitro,and methanol is the optimal substrate among all the substrates investigated.

Objective To prepare and characterize specific monoclonal antibodies( McAbs) against the heavy chain of botulinum neurotoxin serotype A ( BoNT/AHc ) .Methods BALB/c mice were immunized with purified BoNT/AHc protein.After the fusion of mouse splenic cells with SP2/0 cells, hybridoma cell lines secreted McAbs against BoNT/AHc. The McAbs obtained were characterized by indirect ELISA, Western blotting and rapid isotypingassay before being used in ELISA to detect interaction sites in McAbs and BoNT/AHc preliminarily.Results Antigen protein BoNT/AHc of high purification was obtained.Four hybridoma cell lines secreting McAbs against BoNT/AHc were screened,named 1A4,3H3, 3H7 and 5H8,respectively.Their titers of McAbs were all above 3.0 ×103 .They were specifically combined with BoNT/AHc protein by Western blotting.The isotype of 1A4 and 3H7 was IgG1(Κ),that of 3H3 was IgM(Κ),and that of 5H8 was IgG2b(Κ).Additive ELISA showed that epitopes recognized by the four McAbs were close.ELISA analysis confirmed the interaction epitopes in McAbs and BoNT/AHc.Conclusion Monoclonal antibodies against BoNT/AHc are prepared and characterized,providing effective tools for studying the neutralizing antibody and antibody epitopes of BoNT/AHc.

Objective The aim of this study was to investigate the possible effect of glycated low density lipoprotein ( LDL ) on the proliferation, differentiation, and expression of low-density lipoprotein receptor-related protein5(LRP5)mRNA,dickkopf-1(DKK1)mRNAinmouseosteoblasts(MC3T3-E1cells). WhethertheWnt signaling pathway was involved in the above process was explored else. Methods Mouse MC3T3-E1 cells were culturedwithvariousconcentrationsofglycatedLDL(glycatedLDLlevelwas2.4%,5.3%,8.7%,13.9%)for24, 48, 72 h. Proliferation of MC3T3-E1 cell was measured by CCK-8, the osteocalcin level in the medium was determined by ELISA and the expression of LRP5 mRNA, DKK1 mRNA was measured by realtime PCR. Results After cells being incubated with 5. 3% of glycated LDL for 24 h, the inhibition of MC3T3-E1 cells was more marked than that in control group(P<0. 01). The higher glycated LDL level, the severer the inhibition. The effect of LDL on the MC3T3-E1 cell proliferation was time- and dose-dependent under certain conditions. Osteocalcin level in cell culture fluid with glycated LDL was lowered significantly compared with control group. Meanwhile, the expression of DKK1 mRNA was increased significantly but expression of LRP5 mRNA decreased (P<0. 01) in MC3T3-E1 cells cultured with 5. 3% of glycated LDL for 24 h. Conclusions Proliferation and differentiation of the osteoblasts in mice can be inhibited significantly by glycated LDL. The possible mechanism in the role played by glycated LDL on the proliferation and differentiation in MC3T3-E1 cells seems to be related to expression of LRP5 mRNA, DKK1 mRNA in the Wnt signaling pathway.

Objective To clone 2-hydroxyacid dehydrogenase (HADH) gene from Ketogulonigenium vulgare(KGV) Y25 and investigate its expression , purification, and enzymatic characterics .Methods The hadh gene was amplified from ketogulonigenium vulgare Y 25 and cloned into the expression plasmid pITG .The recombinant plasmid was transformed into Escherichia coli BL21(DE3).HADH was then successfully expressed with induction .To explore its enzymatic characteris-tics,HADH was purified by Ni +exchange chromatography .Results HADH constituted more than 50% of the total cell proteins analyzed by SDS-PAGE,with a relative molecular mass of about 35 ×103.With 2-keto-gulonic acid(2-KGA) as substrate, the optimal pH of HADH was at 8.0 ,while the optimal temperature of the purified HADH was at 45℃.Mean-while, such metal ions and chelating agents as Cu 2+, Ca2+, Mg2+, EDTA, and DEPC exerted little effect on enzymatic activities.The maximum initial activity of the enzyme towards 2-KGA reached 27 U/mg, and the Km was calculated as 2.6 mmol/L.The results of in vivo enzyme activity assay showed that HADH could metabolize 2-KGA.Conclusion The HADH gene form Y25 is successfully expressed in E.coli BL21 ( DE3 ) and the enzymatic characteristics of HADH are explored, which will facilitate subsequent studies on sorbose metabolic pathways and sugar acid conversion .

Objective To present our initial experience of using transumbilical multi-stab laparoscopic pyeloplasty(TMLP) as a treatment for the infants younger than 3 months with severe hydronephrosis caused by ureteropelvic junction obstruction (UPJO).Methods Seventy-two infants younger than 3 months with severe hydronephrosis caused by UPJO underwent TMLP from June 2010 to March 2013 in our center.The average age received operation was 34 days (7-87 days).The operative indications included:① prenatal diagnosis of Society of Fetal Urology Grade 4 hydronephrosis; ②ipsilateral differential renal function being less than 40％.Patients were followed up with physical examinations,ultrasound and radionuclide scans.Results The operations were successfully performed in all the 72 patients.Neither conversion nor additional trocars placement was required and there was no intraoperative complication.The average operative time was 75 min (range,53-118 min).The patients were followed up for 6-36 months (mean,12 months).The renal pelvic anteroposterior diameters were reduced and the renal functions were improved (P＜ 0.05).The anastomoses were proved to be patent and the renal parenchymal thickeness increased.In addition,the scars were barely noticeable.Conclusions TMLP for the infants younger than 3 months with severe hydronephrosis caused by UPJO is feasible,safe and less invasive.The cosmetic results are excellent.But the surgery must be operated by rich experienced surgeon.

Objective To summarize the experience of patient positing,port placements setting and robot cart docking in pediatric robot-assisted laparoscopic upper urinary tract operations.Methods From March 2017 to December 2017,140 robot-assisted laparoscopic upper urinary tract procedures were performed in our institution,including 110 cases of pyeloplasty,15 upper pole heminephroureterectomy,12 simple nephrectomy and 3 adrenalectomy.There were 103 males and 37 females with a range age from 1 month to 18 years.The assistant surgeon was adjacent to the instrument nurse,and patients were placed in a supine position with 60°-80° inclination and keep the legs low to the body.Room setup and patient positioning were similar to the traditional laparoscopic surgery.Semi-hidden incision technique was used in 140 patients:the camera port was placed umbilicus,two additional arm ports (one 5 mm and one 8 mm) were placed under direct vision,the 8 mm arm port was placed on the line of a Pfannenstiel incision and the 5 mm arm port was placed below the Xiphoid along the midline.Finally,a 3 or 5 mm assistant port was placed approximately 3 cm lateral to the inferior arm port,the line of a Pfannenstiel incision.Results The average time was (11.5 ± 3.2) min (10.5-16.5 min) from skin incision to robot cart docking completed.All surgeries were successfully completed without open conversion.One patient required an additional assist port for severe adhesion after the previously open surgery,there was no injury to other viscera.Average operative time was (146.9 ± 48.7)min (78-259 min) and average post-operative hospitalization time was (5.7 ± 1.4) d(4-10 d),respectively.There was no visual scar on abdominal 6 weeks postoperatively,and all parents made comments about their satisfaction with the cosmetic appearance.All operations got complete success at a mean follow up of 6 (1-9) months.Conclusions A good room setup,patient positioning and the semi-hidden incision technique port placements are maintaining the safety of the patient,avoiding compression injuries,allowing maximum mobility of the robotic arms,and facilitating a smooth and efficient surgery,and improving post-operative recovery.