Objective:The aim is to establish L-glutamate specific aminotransferase-L-glutamate dehydrogenase coupling 96-well high throughput screening method,which is applied to molecular evolution of aminotransferase WecE from E.coli.Methods:An optical assay for aminotransferase catalytic activity based on aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time.Mutants library of WecE is obtained by sitedirected saturation mutagenesis.Positive mutants can be screened out through 96-well preliminary screening and flask second screening.Results:The target transamination reaction is coupled with L-glutamate dehydrogenase indicative reaction system which consists of 0.5 U/ml enzyme loading and 0.4 mmol/L NADH.A positive mutant Y321F whose catalytic activity increases 3.4 fold compared to that of wild type is screened out in Tyr 321 saturation mutagenesis library of WecE.Conclusion:An accurate high throughput screening method with weak background interference is established.It offers feasible solution for molecular evolution of L-glutamate specific aminotransferase.

Objective To investigate the past participation and their future intention of donation after citizen death (DCD) work of attendees.Methods A questionnaire study was carried out in the 9th Chinese ICU annual congress and local workshops taken part in difference provinces and cities from May 2015 to July 2015.A 12-multiple self choice questionnaires,collecting the demographic data of attendees and analyzing the ratio of DCD awareness and participation,their attitude towards DCD,and future recommendation of DCD development,were facilitated and immediately collected back.Results A total of 512 questionnaires were collected and 489 were available.97.75％ of the attendees have been awareness of DCD and 62.37％ have ever participated in DCD activity.During the whole survey,85.07％ of them expressed their willingness of future DCD activities participation.The current fierce conflict between medical workers and patients was selected to the main barrier of DCD work.The enforcement of DCD population and organ donation law were most recommended to improve donation success.Conclusion It is the fifth year after DCD programme initiation and full implementation since last year.Despite the obvious raise of DCD recognition among ICU staff,there is still long way to go ahead to the era of satisfactory donation rate.The hostility between medical workers and patients is the main barrier for ICU workers for DCD promotion.

Objective To study the impact of various donor hepatectomy techniques on clinical rehabilitation and postoperative liver regeneration on living donor liver transplant (LDLT) donors.Methods The data of 13 consecutive LDLT carried out from May 2006 to May 2011,including the surgical techniques,postoperative liver function,and liver regeneration in the donors were retrospectively studied.Results The donor operations included 8 right hepatectomies without the middle hepatic vein,2 right hepatectomies with the middle hepatic vein and 3 left hepatectomies.Hepatic function and blood coagulation function returned to normal within two weeks of hepatectomy in all the donors.There was no severe complication and no death.There was a significant positive correlation between the donor liver volume as measured preoperatively on CT and the resected liver weight as measured intraoperatively (r=0.838,P＜0.01).The volume of the remnant liver increased soon after transplantation.The liver regenerated significantly faster in right than in lefft liver donors.The remnant liver of the right liver donors with middle hepatic vein preservation grew faster than the right liver donors without middle hepatic vein preservation.However,there was no significant difference in the recovery of the liver function between the three groups.Conclusions Donor hepatectomy is safe.The postoperative liver regeneration is affected by multiple factors including the remnant liver volume and blood supply of the remnant liver.

Objective To analyze the risk factors for the outcomes of recipients after orthotopic liver transplantation using donation after cardiac death (DCD) donors.Method A retrospective study was performed to observe the available clinical data of 60 patients who had receiced hepatic allografts of DCD donors from July 2007 to December 2012 in our hospital and a 3-year follow-up was conducted to investigate outcome.In the patients whose ALT and/or AST levels were more than 1500 U/L within 72 h following surgery,early allograft dysfunction (EAD) was defined.Potential risk fators right before surgery included donor and recipient age,donor ALT AST,TBIL and WIT,and recipient creatine,TBIL,INR,albumin,MELD,BMI and recipient CIT.Kaplan-meier method was used to calculate the cumulative survival rate.Log-rank test and Cox regression model were performed to analyze donors and recipients related risk factors by univariate and multivariate analysis respectively.All statistical data were analyzed by using SPSS 19.0.Results The overall cumulative survival rate of 1 and 3 years was 76％ and 62％ respectively.Donor ALT,AST and WIT,and recipient Cre,MELD,CIT and EAD were significant risk fators in univariate analysis.However,the multivariate analysis revealed that donor WIT was the only independent risk factor affecting survival in our study.Conclusion By identifying and controlling certain characteristics,the outcomes of DCD liver transplant recipients could be dramaticly improved.

BACKGROUND: Tumor recurrence in liver transplant recipients greatly affects prognosis of liver transplantation with hepatocellular carcinoma (HCC). How to prevent tumor recurrence has aroused increasing attention. Arsenious acid chemotherapy is considered effective on treating moderate or advanced liver cancer, but its utilization following liver transplantation remains few. OBJECTIVE: To explore the role of arsenious acid on tumor recurrence in liver transplant patients with primary HCC extending Milan criteria. RESULTS AND CONCLUSION: All patients were routinely followed up for 3-32 months. Thirty recipients were presented with tumor recurrence, 16 in the chemotherapy group and 14 in the non-chemotherapy group. Tumor recurred in lung, liver graft and bones in most cases. The total recurrence rate was similar in these two groups, but chemotherapy could delay recurrence after transplantation (P=0.026). There was no significance in 6-month, 1-year survival rate between two groups, but the 2-year survival in the chemotherapy group was higher (P=0.037); 6-month tumor-free survival rates in the two groups had no significance, 1-year and 2-year tumor-free in the chemotherapy group were significantly higher than those in the non-chemotherapy group (P=0.030, 0.023). Intravenous arsenious acid chemotherapy can delay tumor recurrence and prolong survival in liver transplant patients with HCC extending Milan criteria.

Objective To evaluate the efficacy of liver grafts with warm ischemia and with different cold preservation time in liver transplantation.Methods The clinical data of 154 patients who received liver transplantation at the First Affiliated Hospital of Sun Yat-sen University from January 2006 to December 2007 were retrospectively analyzed.The warm ischemia time of the liver grafts obtained from the non-heart-beating donors was within 10 minutes.According to cold perservation time of the liver grafts,patients were divided into 3 groups:the cold preservation time of the liver grafts was within 8 hours,8-12 hours and above 12 hours in group I(n=58),group Ⅱ(n=62)and group Ⅲ(n=34),respectively.The peak level of alanine aminotransferase(ALT),primary graft dysfunction(PGD)after liver transplantation,acute rejection response,biliary complications,vessel complications,perioperative infections and the survival of liver grafts and recipients among the 3 groups were analyzed via chi-square test,t test and variance analysis.Results No PGD was detected in the 3 groups after liver transplantation.All patients were followed up for 8-32 months.The peak level of ALT,incidence of infection and biliary complication,survival of liver grafts and recipients were(482±357)U/L,12%(7/58),12%(7/58),86%(50/58)and 88%(51/58)in group Ⅰ,and were(1274±608)U/L,29%(10/34),26%(9/34),68%(23/34)and 71%(24/34)in group Ⅲ,with significant difference between the 2 groups(t=5.23,X~2=4.28,6.77,4.51,4.28,P<0.05).The peak level of ALT in group Ⅱ was(953±424)U/L,which was significant higher than(482±357)U/L in group Ⅰ(t=4.76,P＜0.05).Conclusions Liver grafts with a warm ischemia time shorter than 10 minutes could tolerate the injury caused by cold preservation with the maximum time of 12 hours.The incidences of biliary complications and postoperative infections are significantly increased and the survivals of liver grafts and recipients are decreased when the cold preservation time exceeds 12 hours.

BACKGROUND:Displacement of the distal fracture fragment is one of the most important facts that lead to cubitus varus fol owing pediatric supracondylar humeral fracture. Mainstream technique emphasized the restoration of posterior-ulnar deviation of the distal fragment. However, there is an absence of supportive evidences from biomechanical studies. OBJECTIVE:To establish models of extension-ulnar type of supracondylar humeral fracture and investigate the mechanical stability of reduced fracture with residual displacements within functional restoration standard, so as to provide mechanic evidences supporting the empirical rule of manipulative reduction-“better anterior than posterior, better radial than ulnar”. METHODS:The fresh cadaveric bone of right upper extremity from a 7-year-old child was scanned using CT. Models of supracondylar humeral fracture differing in contact area of the fracture site and displacement direction of the distal fragment were established and underwent loading tests. Stress in both anterior and posterior margin of the fracture site and Baumann angle were recorded, and data were analyzed and compared. RESULTS AND CONCLUSION:In comparison of stress in the posterior margin, the value was significantly greater in the posteromedial-displacement group than the others. Stress value in fracture with 75%contact area was significantly greater than the other three groups. In comparison of stress in the anterior margin, a significantly greater value was obtained in the posteromedial-displaced group. Stress value in fracture with 85%contact area was significantly greater. When comparing stress in posterior margin and anterior margin, the absolute increment of stress value was greater in posterior displacement group than in anterior displacement group. Baumann angle increased significantly when fragment displaced medial y. Above findings indicated that displacement direction altered the location of stress concentration. Stress augmentation was greater in posterior displacement group. Stress in related area significantly increased constantly when contact area of the fracture site reduced. Baumann changed obviously when fragment displaced medial y. The results preliminarily verify the hypothesis that displacement of the distal fragment was the main contributor to cubitus varus fol owing supracondylar humeral fracture. These findings provided certain evidences supporting the empirical rule“better anterior than posterior, better radial and ulnar”.

Objective To evaluate the effect of endoscopic retrograde cholangiopancreatography (ERCP) in treatment of biliary complications after liver transplantation. Methods Data of 39 patients who underwent ERCP between January 2005 and December 2007 because of biliary complications after liver trans-plantation were retrospectively evaluated. Endoscopic sphincterotomy, dilatation, nasal-biliary drainage (ENBD) and stent placement were performed in 25 patients with biliary strictures (14 at anastomosis site and 11 at non-anastomosis site). ENBD and stent placement were applied in 6 patients with biliary leakage, while endoscopic sphincterotomy, ENBD and stone extraction with baskets were performed in 16 patients with biliary stones. Procedures were repeated when necessary. Results ERCP was successfully performed at a rate of 95.9% (94/98) without any severe complications. Strictures at anastomosis site were resolved in all patients (100%, 14/14), while for strictures at non-anastomosis site, only 27.3% (3/11) were cured. Biliary leakage was resolved in 83.3% (5/6) patients. Complete bile duet clearance was achieved in 81.3% (13/16) of the patients with biliary stones. Conclusion ERCP proves to be safe and effective in the treatment of post liver transplantation biliary complications with low incidence of severe complications.

Objective To evaluate the diagnosis and treatment of early-stage hepatic artery thrombosis(HAT) after adult liver transplantation.Methods387 consecutive adult patients who underwent liver transplantation from June 2007 to October 2010 by the same surgery team in the Transplant Center,First Affiliated Hospital of Sun Yat-sen University were retrospectively studied.Hepatic arterial blood flow was monitored by color Doppler ultrasound (DUS) daily during the first week after transplantation.Ultrasonic contrast or hepatic artery angiography was performed on recipients with suspected HAT.Results10 patients developed HAT on 7(2-18)d after operation.The incidence of HAT was 2.6％ (10/387).Interventional therapy was performed in 2 patients with one patient who received a stent because of hepatic artery stricture.Three patients underwent emergent hepatic artery revascularization combined with intra-arterial urokinase thrombolysis treatment.One developed a rethrombosis and died.The remaining 2 patients received re-transplantation.Three patients died of liver failure and severe infection.The mortality rate was 40％ (10/387).ConclusionsIt is essential to diagnoses HAT by monitoring the artery flow by Doppler ultrasound screening in the early period after operation.Interventional therapy,emergent hepatic artery revascularization and re-transplantation are effective rescue treatments.Prevention of HAT is most important.

Objective To investigate the effects of ischemic preconditioning on the cholesterol content and the activity of Na+-K+-ATPase of hepatocytes following cold preservation in rats.Methods Twenty-five rats were randomly divided into five groups,including control group (C),cold preservation group (Ⅰ),ischemic preconditioning group (ⅠP),atorvastatin (30 μmol/L) treatment group (A30),and atorvastatin (100 μmol/L) treatment group (A100).The cholesterol content and the activity of Na+ -K+ -ATPase were assessed.Results The cholesterol contents on the rat liver tissue cell membrane in the C group,Ⅰ group,ⅠP group,A30 group and A100 group were (310.4 ± 27.5),(187.7±13.1),(394.3±25.9),(201.8±14.6) and (122.6±7.7) nmol/mg protein,and activity of the Na+ -K+ -ATP enzyme was (46.55 ± 3.20),(27.4 ± 2.81),(52.71 ± 3.02),(30.67 ±2.78) and (19.64 ± 2.11) μmol Pi/hr mg protein,respectively (P＜0.05).There was no significant difference in the plasma membrane phospholipid content among the five groups (P＞0.05).Conclusion Reduction of cholesterol content and Na+ K+ -ATPase activity on the liver cytoplasmic membrane is one of the factors causing donor liver cold preservation injury,but ischemic preconditioning can significantly improve cell membrane Na+ -K+ -ATPase activity and increase cytoplasmic membrane cholesterol content. Use of atorvastatin statins can reduce cytoplasmic membrane cholesterol synthesis,and significantly decrease Na+ -K+ -ATPase activity,thereby alleviating the donor liver cold preservation injury.