Objective To express virus-like particles(VLP) of enterovirus 71 (EV71) in Han-senula polymorpha.Methods The coding sequences of P1 and 3CD genes of EV71 were optimized accord-ing to codon usage bias of Hansenula polymorpha for achieving high expression , and then cloned into the ex-pression vector PMV of Hansenula polymorpha .The recombinant expression vector PMV-P1-3CD was trans-formed into Hansenula polymorpha AU 0501 .The transformants were stably cultured in selective medium (Yeast Nitrogen Base) and screened for strains with positive P1 and 3CD genes by PCR.Then an induced cultivation on the recombinant strains were performed in a medium supplemented with methanol to a final concentration of 1.0%and the expressed products were analyzed by SDS-PAGE and Western blot assays to select high expression strains .The high expression strains were cultured in 30 L fermentor and its fermenta-tion products were analyzed by electronic microscope after purification .Results EV71 recombinant expres-sion strains were successfully constructed .The results of SDS-PAGE showed that the expressed products had obvious VP3, VP1, VP0 protein bands with molecular weights of 26×103, 33×103 and 35×103, respective-ly, which were consistent with the expected molecular weight of the fusion proteins .Western blot demonstra-ted that the expressed products could be specifically recognized by the polyclonal antibody against EV 71-VP1 at 33 ×10 3 , indicating its high immunoreactivity .ELISA confirmed that the expression level of EV 71 fermen-tation products was reached to 200 mg/L.Electronic microscope analysis showed that the VLP of recombi-nant EV71 were 24-30 nm in diameter with normal structure .Conclusion The virus-like particles of human enterovirus 71 are successfully expressed in Hansenula polymorpha , which provides a foundation for the fur-ther development of EV 71 VLP vaccine .

Objective To evaluate the therapeutic effect of different types of biliary tract reconstruction on the post- resection of congenital choledochal cyst (CCC). Methods 106 cases of CCC undergoing resection of CCC with Roux-en-Y hepaticojejunostomy as biliary tract reconstruction were followed up and analysed. Results In this series, three kinds of biliary tract reconstruction were performed, including single Roux-en-Y hepaticojejunostomy in 48 cases, intussusceptive valve and rectangular valve lying on the jejunal segmental of Roux-en-Y hepaticojejunostomy in 37 and 21 cases, respectively. 61 cases were followed up for 4.82 years in average. None of them occurred anastomasis stricture. There were 4 cases with ascending cholangitis after primary operation: of them 3 after single Roux-en-Y hepaticojejunostomy; symptoms of 2 out of the 3 cases disappeared after reoperation to set up an intussusceptive valve plasty on the jejunal segmental of Roux-en-Y hepaticojejunostomy; another one was in rectangular valve group. Occasional abdominal pain tooke place in 8 patients, of them, 5 in single Roux-en-Y hepaticojejunostomy group, 2 and 1 in intussusceptive valve and rectangular valve groups, respectively. No patients suffered from ascending cholangitis in the intussusceptive valve plasty group. Conclusions The postoperative ascending cholangitis and anastomasis stricture can be prevented effectively, if a prophylactic intussusceptive valve on the jejunal segmental of Roux-en-Y hepaticojejunostomy is carried out after resection of CCC for biliary tract reconstruction. This procedure corresponds to the biliary tract physiology.

Objective To evaluate the efficacy and safety of oral propranolol versus atenolol in the treatment of infantile hemangioma(IH). Methods A total of 75 infants with IH aged 5-24 weeks were randomly divided into two groups: propranolol group(n = 30)orally administrating propranolol 2 mg · kg?1 · d?1 in 3 divided doses daily for 24 consecutive weeks, atenolol group(n=45)orally administrating atenolol 1 mg · kg?1 · d?1 once a day for 24 consecutive weeks. After 1?, 4?, 12?, 24?week treatment, the infants with IH were followed and adverse reactions were recorded. In addition, the activity of IH was assessed by hemangioma activity score(HAS)before and after 24?week treatment, and changes of HAS were compared between the propranolol group and atenolol group. Results There was no significant difference in the proportion of patients experiencing satisfactory regression of hemangioma between the propranolol group and atenolol group(70%[21/30]vs. 75.6%[34/45], P>0.05). Treatment failure occurred in one patient in the propranolol group because of severe airway hyperreactivity, and in another patient in the atenolol group because of drug resistance. The incidence rates of gastrointestinal reactions, central nervous system adverse effects, chills on the extremities and bronchiolitis complicated by airway hyperreactivity were all significantly higher in the propranolol group than in the atenolol group(all P<0.05). None of hypotension, hypoglycemia and bradycardia occurred in the two groups. Conclusion Compared with propranolol, atenolol shows similar efficacy but less adverse effects in the treatment of IH.

AIM: To explore the expressive role of lipoprotein-associated phospholipase A_2, high sensitive C-reactive protein and matrix metalloproteinase-9 in vulnerable atherosclerotic plaques in a rabbit model. METHODS: Forty eight New Zealand white male rabbits were randomly divided into 4 groups (12 rabbits each): control group, stable plaque group, p53 group, and p53+drug group. Rabbits in control group were fed with a regular diet and underwent sham operation. Rabbits in stable plaque group, p53 group and p53+drug group underwent balloon induced arterial wall injury and then were fed on a diet with 1% cholesterol. The animals were all fed for 3 months, then the rabbits in p53 group and p53+drug group underwent Ad5-CMV p53 transfection at 10th week. Before killed, the animals in p53+drug group underwent pharmacological triggering with Russell's viper venom (RVV) and histamine to induce the rupture of the atherosclerotic plaques. At the 1st day and before sacrifice, the serum was collected for measuring Lp-PLA_2, hs-CRP, MMP-9, HDL, LDL and VLDL. The expressions of Lp-PLA_2, hs-CRP and MMP-9 in tissues were determined by the methods of hybridization and immunohistochemistry. RESULTS: At the end of 12th week, the serum and tissue levels of Lp-PLA_2 and MMP-9 in stable plaque group, p53 group and p53+drug group were significant different from those in control group and in each group at the first day (P<0.05). The serum levels of Lp-PLA_2 and hs-CRP in p53 group and p53+drug group were significantly higher than those in control group and stable group (P<0.05). The serum levels of Lp-PLA_2, hs-CRP and MMP-9 were all significantly different between p53 group and p53+drug group (P<0.05). At the end of 12th week, pathological results showed that 4 groups were normal artery, stable plaque, vulnerable plaque and rupture plaque, respectively. The fabric cap was thicker in plaque groups than that in normal group (P<0.05). The rupture and formation of thrombus were more significant in p53+drug group than those in p53 group. The serum level of Lp-PLA_2 had negative interrelated relationship with fabric cap in plaque groups (r=-0.710, P<0.01), and hs-CRP, MMP-9 had no interrelated relationships with fabric cap in plaque groups. CONCLUSION: Base on the successful establishment of the atherosclerotic plaque animal model, serum Lp-PLA_2 shows better interrelated relationships to plaques stability. Combination with hs-CRP and MMP-9, we can exactly evaluate the nature of plaques.

Objective To investigate the influence factors of tumor diameter and related prognostic factors on the prognosis of hilar cholangiocarcinoma.Methods The retrospective case-control study was conducted.The clinicopathological data of 240 patients who underwent resection of hilar cholangiocarcinoma in the West China Hospital of Sichuan University between January 1995 and January 2013 were collected,including 104 patients with tumor diameter ≤ 2 cm (8 with tumor diameter ≤ 1 cm and 96 with 1 cm ＜ tumor diameter ≤ 2 cm),85 with 2 cm ＜ tumor diameter ≤ 3 cm and 51 with tumor diameter ＞ 3 cm (40 with 3 cm ＜ tumor diameter ≤ 4 cm and 11 with tumor diameter ＞ 4 cm).Observation indicators:(1) surgical situations;(2) follow-up situations;(3) risk factors analysis affecting the prognosis of patients;(4) correlation analysis between related prognostic indicators and tumor diameter.The follow-up using outpatient examination and telephone interview was performed to detect the survival up to August 2016.The survival curve and survival rate were respectively drawn and calculated by the Kaplan-Meier method,and the Log-rank test was used for survival analysis.The prognostic factors and correlation between related prognostic indicators and tumor diameter were respectively analyzed using the COX proportional hazard model and logistic regression model.Results (1) Surgical situations:240 patients underwent successful resection of hilar cholangiocarcinoma and lymph node dissection.Of 73 patients with postoperative complications,1 died of intraperitoneal infection induced to systemic infection and multiple organ failure,1 diel of renal failure,and other patients were cured by symptomatic treatment.(2) Follow-up situations:240 patients were followed up for 12.0-98.0 months,with a median time of 47.4 months.The overall median survival time,1-,3-and 5-year overall survival rates were respectively 30.6 months,81％,47％ and 29％.The median survival time and 5-year survival rate were 46.5 months,34％ in patients with tumor diameter ≤ 2 cm and 30.5 months,30％ in patients with 2 cm ＜ tumor diameter ≤ 3 cm and 13.8 months,20％ in patients with tumor diameter ＞ 3 cm,respectively,with a statistically significant difference (x2 =17.83,P＜0.05).Results of further analysis showed the median survival time and 5-year survival rate were 31.3 months,38％ in patients with tumor diameter ≤ 1 cm and 46.5 months,34％ in patients with 1 cm ＜ tumor diameter ≤ 2 cm,respectively,with no statistically significant difference (x2=1.16,P＞O.05).The median survival time and 1-year survival rate were 14.7 months,62％ in patients with 3 cm ＜ tumor diameter ≤ 4 cm and 13.0 months,55％ in patients with tumor diameter ＞ 4 cm,respectively,with no statistically significant difference (x2 =2.34,P＞O.05).(3) Risk factors analysis affecting the prognosis of patients:univariate analysis showed that tumor diameter,surgical margin,lymph node metastasis,vascular invasion and histological differentiation were the related factors affecting patients' prognosis [hazard ratio (HR)=1.456,8.714,1.737,2.246,1.665;95％ confidence interval (C I):1.212-1.748,5.558-13.663,1.311-2.301,1.494-3.378,1.375-2.016,P ＜ 0.05].The multivariate analysis showed that 2 cm ＜ tumor diameter ≤ 3 cm,tumor diameter ＞ 3 cm,R1 resection,lymph node metastasis and low-differentiated tumor were the independent risk factors affecting poor prognosis of patients (HR =1.559,1.868,7.410,1.521,2.274,95％ CI:1.125-2.160,1.265-2.759,4.497-12.212,1.136-2.037,1.525-3.390,P＜0.05).(4) Correlation analysis between related prognostic indicators and tumor diameter:the results of univariate analysis showed that there was a correlation between lymph node metastasis,vascular invasion,histological differentiation and T staging of American Joint Committee on Cancer (AJCC) and tumor diameter of 2 cm as a cut-off point (x2 =6.063,4.950,8.770,9.069,P＜0.05).There was a correlation between surgical margin,lymph node metastasis,vascular invasion and histological differentiation and tumor diameter of 3 cm as a cut-off point (x2=10.251,9.919,5.485,15.632,P＜0.05).The results of multivariate analysis showed that lymph node metastasis and T staging of AJCC were independent related factors affecting tumor diameter of 2 cm as a cut-off point[odds ratio (OR) =1.882,2.104,95 ％CI:1.075-3.293,1.220-3.631,P＜0.05];surgical margin and lymph node metastasis were independent related factors affecting tumor diameter of 3 cm as a cut-off point (OR=3.187,2.211,95 ％CI:1.377-7.379,1.133-4.314,P＜0.05).Conclusions The 2 cm ＜ tumor diameter ≤ 3 cm,tumor diameter ＞ 3 cm,R1 resection,lymph node metastasis and low-differentiated tumor are the independent risk factors affecting the prognosis of patients with hilar cholangiocarcinoma.Three cm (T staging in De Oliveira staging system) as the second cut-off point is feasible,meanwhile,2 cm cut-off point may be become another potential tumor dividing point described in De Oliveira staging system.

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.
Butanols ; pharmacology ; Cell Engineering ; methods ; Clostridium acetobutylicum ; cytology ; drug effects ; genetics ; physiology ; DNA Methylation ; genetics ; DNA Mismatch Repair ; genetics ; Evolution, Molecular ; Genome, Bacterial ; genetics ; MutS DNA Mismatch-Binding Protein ; genetics ; Mutation ; Operon ; genetics ; Stress, Physiological ; drug effects ; genetics

Objective:To establish autoverification rules for coagulation tests in multicenter cooperative units, in order to reduce workload for manual review of suspected results and shorten turnaround time (TAT) of test reports, while ensure the accuracy of results.Methods:A total of 14 394 blood samples were collected from fourteen hospitals during December 2019 to March 2020. These samples included: Rules Establishment Group 11 230 cases, including 1 182 cases for Delta check rules; Rules Validation Group 3 164 cases, including 487cases for Delta check; Clinical Application Trial Group 77 269 cases. Samples were analyzed for coagulation tests using Sysmex CS series automatic coagulation analyzers, and the clinical information, instrument parameters, test results, clinical diagnosis, medication history of anticoagulant and other relative results such as HCT, TG, TBIL, DBIL were summarized; on the basis of historical data, the 2.5 and 97.5 percentile of all data arranged from low to high were initially accumulated; on the basis of clinical suggestions, critical values and specific drug use as well as relative guidelines, autoverification rules and limits were established.The rules were then input into middleware, in which Stage I/Stage II validation was done. Positive coincidence, negative coincidence, false negative, false positive, autoverification pass rate, passing accuracy (coincidence of autoverification and manual verification) were calculated. Autoverification rules underwent trial application in coagulation results reports.Results:(1) The autoverification algorisms involve 33 rules regarding PT/INR, APTT, FBG, D-dimer, FDP,Delta check, reaction curve and sample abnormalities; (2)Autoverification Establishment Group showed autoverification pass rate was 68.42% (7 684/11 230), the false negative rate was 0%(0/11230), coincidence of autoverification and manual verification was 98.51%(11 063/11 230), in which positive coincidence and negative coincidence were respectively 30.09% (3 379/11 230) and 68.42%(7 684/11 230); Autoverification Validation Group showed autoverification pass rate was 60.37%(1 910/3 164), the false negative rate was 0%(0/11 230), coincidence of autoverification and manual verification was 97.79%(3 094/3 164), in which positive coincidence and negative coincidence were respectively 37.42%(1 184/3 164) and 60.37%(1 910/3 164); (3) Trialed implementation of these autoverification rules on 77 269 coagulation samples showed that the average TAT shortened by 8.5 min-83.1 min.Conclusions:This study established 33 autoverification rules in coagulation tests. Validation showedthese rules could ensure test quality while shortening TAT and lighten manual workload.

Objective: To investigate the clinical value of lymph node dissection (LND) in the radical resection of intrahepatic cholangiocarcinoma (ICC). Methods: The propensity score matching and retrospective cohort study was conducted. The clinicopathological data of 448 patients with ICC who were admitted to 12 medical centers from December 2011 to December 2017 were collected, including 279 in the Eastern Hepatobiliary Surgery Hospital of Navy Medical University, 32 in the Mengchao Hepatobiliary Hospital of Fujian Medical University, 21 in the First Hospital Affiliated to Army Medical University, 20 in the Cancer Hospital Chinese Academy of Medical Science and Peking Union Medical College, 19 in the West China Hospital of Sichuan University, 18 in the Second Hospital Affiliated to Zhejiang University School of Medicine, 18 in the Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 16 in the Beijing Friendship Hospital Affiliated to Capital Medical University, 10 in the Xuanwu Hospital Affiliated to Capital Medical University, 7 in the Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, 5 in the Beijing Tiantan Hospital Affiliated to Capital Medical University, and 3 in the Affiliated Hospital of North Sichuan Medical College. There were 281 males and 167 females, aged from 22 to 80 years, with a median age of 57 years. Of the 448 patients, 143 with routinely intraoperative LND were divided into LND group and 305 without routinely intraoperative LND were divided into control group, respectively. Observation indicators: (1) the propensity score matching conditions and comparison of general data between the two groups after matching; (2) intraoperative and postoperative situations; (3) follow-up; (4) survival analysis. Patients were followed up by outpatient examination, telephone interview and email to detect survival of patients and tumor recurrence up to October 31, 2018 or death. Follow-up was conducted once every 3 months within postoperative 1-2 years, once every 6 months within postoperative 2-5 years, and once a year after 5 years. The propensity score matching was realized using the nearest neighbor method with 1∶1 ratio. Measurement data with normal distribution were represented as Mean±SD, and comparison between groups was analyzed using the t test. Measurement data with skewed distribution were represented as M (range), and comparison between groups was analyzed using the Mann-Whitney U test. Count data were described as absolute numbers, and comparison between groups was analyzed using the chi-square test or Fisher exact probability. Kaplan-Meier method was used to calculate survival rates and draw survival curve, and Log-rank test was used to perform survival analysis. Univariate analysis and multivaraiate analysis were conducted using the Log-rank test and COX regression model, respectively. Results: (1) The propensity score matching conditions and comparison of general data between the two groups after matching: 286 of 448 patients had successful matching, including 143 in each group. The number of males and females, cases with hepatitis, cases with grade A and B of Child-Pugh classification, cases with distance from margin to tumor <1 cm and ≥1 cm, cases with highly, moderately, and poorly differentiated tumor, cases with anatomical hepatectomy and nonanatomical hepatectomy before propensity score matching were 77, 66, 33, 96, 47, 70, 73, 105, 38, 79, 64 in the LND group, and 204, 101, 121, 165, 140, 207, 98, 251, 54, 124, 181 in the control group, respectively, showing significant differences in the above indices between the two groups (χ²=7.079, 11.885, 7.098, 14.763, 5.184, 8.362, P<0.05). After propensity score matching, the above indices were 77, 66, 33, 96, 47, 70, 73, 105, 38, 79, 64 in the LND group, and 79, 64, 29, 88, 55, 71, 72, 112, 31, 74, 69 in the control group, respectively, showing no significant difference in the above indices between the two groups (χ²=0.056, 0.329, 2.158, 0.014, 0.936, 1.400, P>0.05). (2) Intraoperative and postoperative situations: the operation time, volume of intraoperative blood loss, cases with intraoperative blood transfusion, cases with postoperative complications, and duration of postoperative hospital stay were 265 minutes (range, 160-371 minutes), 300 mL (range, 200-500 mL), 37, 46, 12 days (range, 9-17 days) for the LND group, and 59 minutes (range, 46-250 minutes), 200 mL (range, 100-400 mL), 24, 25, 9 days (range, 7-11 days) for the control group, respectively. There was no significant difference in the volume of intraoperative blood loss or cases with intraoperative blood transfusion between the two groups (Z=1.700, χ²=3.520, P>0.05). There were significant differences in the operation time, cases with postoperative complications, and duration of postoperative hospital stay (Z=6.520, χ²=8.260, Z=4.270, P<0.05). (3) Follow-up: 143 patients in the LND group was followed up for 18-26 months. The 1-, 3-, 5-year survival rates were 66.9%, 32.8%, 25.0%, and the median survival time was 22.0 months (range, 18.0-26.0 months). In the control group, 143 patients were followed up for 11-32 months. The 1-, 3-, 5-year survival rates were 71.7%, 38.0%, 31.0%, and the median survival time was 22.0 months (range, 11.0-32.0 months). There was no significant difference in the overall survival between the two groups (χ²=0.466, P>0.05). (4) Survival analysis: results of univariate analysis showed that distance from margin to tumor, tumor diameter, the number of tumors, microvascular tumor thrombus, and lymph node metastasis by pathological examination were related factors for prognosis of patients undergoing radical resection of ICC (χ²=10.662, 9.477, 16.944, 6.520, 11.633, P<0.05). Results of multivariate analysis showed that distance from margin to tumor<1 cm, tumor diameter>5 cm, multiple tumors, microvascular tumor thrombus, positive lymph node metastasis by pathological examination were independent risk factors for prognosis of patients undergoing radical resection of ICC (hazard ratio=0.600, 1.571, 1.601, 1.750, 1.723, 95% confidence interval: 0.430-0.837, 1.106-2.232, 1.115-2.299, 1.083-2.829, 1.207-2.460, P<0.05). Conclusion Lymph node metastasis is an independent risk factor affecting the prognosis of ICC patients, and it is necessary to perform LND in radical resection for patients with ICC and lymph node metastasis.