In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.
Clone Cells ; Mutagenesis, Site-Directed ; Mutation ; Plasmids/genetics* ; Polymerase Chain Reaction/methods*

OBJECTIVETo summarize the clinical features, biochemical change and genetic mutations of a neonate with congenital bile acid synthesis disorder type 2.

METHODSClinical features, blood biochemical index, gene analysis and treatment of the patient were reviewed.

RESULTSThe patient presented with the symptoms of jaundice 3 days after birth but without skin itching. Pale stool was noted. Subsequently, he presented with hepatomegaly, blood coagulation disorders, left cochlear nerve damage, liver cirrhosis and remarkable growth retardation. Serum biochemistries showed that bilirubin and transaminase were elevated, while γ -GT and total bile acid was normal. Abdominal ultrasonography indicated decline of gallbladder contraction. Cholangiography showed normal extra- and intrahepatic bile ducts and patent biliary tract. Liver biopsy showed intrahepatic cholestasis. Gene testing has identified a homozygous mutation in AKR1D1 gene.

CONCLUSIONCongenital bile acid synthesis disorder should be suspected when a neonate has presented with jaundice, elevated bilirubin and transaminase, normal or reduced TBA and γ -GT. Genetic testing and urine mass spectrometry analysis can diagnose congenital bile acid synthesis disorder. Early therapy is crucial to patients with congenital bile acid synthesis disorder.

Objective To investigate the clinical efficacy of primary closure in laparoscopic common bile duct exploration (LCBDE).Methods The retrospective cross-sectional study was conducted.The clinicopathological data of 2 429 patients who underwent primary closure in LCBDE in the Second Hospital of Chengdu City from March 1992 to December 2017 were collected.Patients underwent laparoscopic cholecystectomy (LC) + stone extraction using LCBDE or extracorporeal shock wave lithotripsy (ESWL),laparoscopic endoscopic sphincteropapillotomy (LEST) was performed selectively,then underwent laparoscopic endoscopic nasobiliary drainage (LENBD) or laparoscopic transabdominal antegrade-guide common bile duct stent implantation,finally underwent primary closure of common bile duct.Observation indicators:(1) surgical situations;(2) postoperative recovery;(3) postoperative short-term complications;(4) follow-up situation.Follow-up using outpatient examination and telephone interview was performed to observe long-term surgical complications once every 3 months up to 1 year postoperatively.Measurement data with normal distribution were represented as (x) ± s.Measurement data with skewed distribution were described as M (range).Results (1) Surgical situations:of 2 429 patients,2 251 underwent successful stone extraction using LCBDE,relief of the obstruction and primary closure of common bile duct,with depletion of stones;15 underwent stone extraction using conversion to open surgery,with depletion of stones;163 had residual stones or surgery-related complications.Of 2 429 patients,1 144,898,223,110 and 54 were respectively detected in grade N or 0,1,2,3 and 4 of laparoscopic distal of the common bile duct smooth classification (LDSC);599 underwent LEST,367 underwent LENBD,207 indwelled urinary catheter through cystic duct stump,125 underwent laparoscopic transabdominal antegrade-guide common bile duct stent implantation and 1 131 underwent primary closure in LCBDE only.Number of removing the stones,diameter of common bile duct,volume of intraoperative blood loss and operation time was (2.5±0.2)per case,(0.7±0.4)cm,(22.4±2.6)mL and (100±12) minutes,respectively.(2) Postoperative recovery:time of postoperative gastrointestinal function recovery,duration of hospital stay and treatment expenses were respectively (2.5±0.5) days,(7.3± 1.2) days and (2.7±0.3) × 104 yuan.Of 2 429 patients,367 removed nasobiliary catheter at 3-7 days postoperatively,207 removed urinary catheter at 3-6 weeks postoperatively,and 125 removed common bile duct stent through duodenoscope port at 1-4 months postoperatively.(3) Postoperative short-term complications:incidence of postoperative short-term complications in 2 429 patients was 6.711％ (163/2 429).① Ninety-four patients with bile leakage were cured after drainage and symptomatic and supportive treatment.② Of 29 patients with residual stones:25 with residual stones of common bile duct were cured by stone extraction using endoscopic sphincterotomy of duodenal papilla,and 4 with residual stones of intrahepatic bile duct didn't receive treatment.③ One patient died at 15 days after surgery for pancreatic cancer.④ Of 39 with postoperative other complications:2 with postoperative hemorrhage were cured by laparoscopic reoperation;12,19 and 3 were respectively complicated with postoperative inflammatory stenosis of duodenal papilla induced to short-term obstructive jaundice,mild acute pancreatitis and stress ulcer bleeding of upper digestive tract,and they were improved by endoscopy or non-operation treatment;2 with stenosis of bile duct didn't receive treatment;1 had a miss ligation at bending section of front zone of nasobiliary catheter,and ligation was removed by endoscopic retrograde cannulation of the pancreatic at 19 days postoperatively.(4) Follow-up situation:of 2 429 patients,1 749 were followed up for 3-12 months,with a median time of 6 months.During the follow-up,of 1 749 patients,2 had mild stenosis in the primary closure area of common bile duct incision and 1 had stenosis of duodenal papilla,they were not treated,and other patients didn't have related complications.Conclusion Controlling strictly the operative indication,primary closure of common bile duct in LCBDE is safe and feasible,with satisfactory clinical outcomes.

Objective To analyze our experience in primary closure of common bile duct after laparoscopic bile duct exploration.Methods From June 1992 to March 2018,2 740 patients underwent primary closure of common bile duct after laparoscopic common bile duct exploration in the Second People's Hospital of Chengdu.Results The operations were successfully carried out in 2 534 (92.4％) out of 2 740 patients,of whom 15 patients (0.6％) were converted to open common bile duct exploration.Bile leakage occurred in 113 patients (4.1％).Residual stones were found in 29 patients (1.1％).One patient (0.1％) who had a pancreatic carcinoma died on postoperation day 15.48 patients (1.8％) developed other complications.The total postoperative complication rate was 7.0％ (191/2 740).Conclusion In suitable patients,laparoscopic bile duct exploration with primary closure was feasible,safe and effective.

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

To explore the operation methods and indications of the duodenoscopic papillotomy (IEST) with endoscopic nasobiliary drainage (IENBD) for the treatment of duodenal papilla stenosis during the course of common bile duct operation.The clinical data of 219 cases of cholecystolithiasis with choledocholith and the stenosis of papillary underwent endoscopic sphincterotomy (IEST) plus endoscopic nasobiliary drainage (IENBD) in the Second People's Hospital of Chengdu were retrospectively analyzed.It was successful in 198 cases who had the gallbladder and common bile duct stones removed,and endoscopic papillary dissection was performed and the nasobiliary tube was successfully inserted.Nasobiliary drainage was successful in 186 cases (93.9％) of 198 cases.No liquid outflow was observed in nasobiliary drainage in 7 cases (3.5％).Nasal bile duct slipped early in 5 case (2.5％).Primary closure of bile duct incision was completed in 198 cases.It failed in 4 cases (2.0％) who had the bile leakage with primary closure of duct incision.Mild pancreatitis after operation occurred in 3 cases (1.5％).Nose bile duct ligation was performed in 1 case (0.5％).The overall postoperative complication rate was 4.0％ (8/198).IEST + IENBD in open laparotomy was successful in 21 cases.No perforation of intestine and bile duct,bleeding,severe pancreatitis and other complications and death were detected postoperatively in two groups.During the course of laparoscopy and open laparotomy,IEST + IENBD in treating cholecystolithiasis with choledocholith and the stenosis of papillary and primary closure of duct incision after the endoscopic nasobiliary drainage is safe and effective.

Objective To observe the curative effect of lumbar sympathetic nerve injury combined with systemic and local medication in intractable lower limb ischemic pain.Methods21 patients with intractable lower limb ischemic pain accepted lumbar sympathectomy,at the same time foot topical Votalinemulgel used and Combined with anticonvulsants and opioids according to circumstances,the visual analogue scale of resting pain(visual analogue scale,VAS),the Local skin temperaturebefore and after treatment were observed and evaluated.ResultsAfter Lumbar sympathetic nerve block and topical Votalinemulgel,21 patients had pain relief.After treatment, 2 patients had poor effect,the remaining 19 patients had pain relief to VAS<3, 4 patients did not need systemic analgesic drugs,the rest of the patients need systemic analgesic drugs.ConclusionIt is necessary to adopt multimodal comprehensive treatment for intractable lower limb ischemic pain.

Objective To evaluate the clinical efficacy of modified Chen's biliojejunostomy technique in treating hilar cholangiocarcinoma.Methods The clinical data of the patients with hilar cholangiocarcinoma from January 2011 to June 2014 in the PLA general hospital and its first affiliated hospital were retrospective ly studied,and 17 of them underwent modified biliojejunostomy.There were 10 male and 7 female patients with a mean of 65 years old (range 34 ～82).Cases diagnosed as Bismuth-Corlette Type Ⅱ,Ⅲ a,Ⅲ b and Ⅳ were 2,6,4,and 5,respectively.Results Liver segment Ⅳ were resected from 2 patients,segment Ⅳ + Ⅴ from 7 patients and segment Ⅳ + Ⅴ + Ⅰ from 8 patients.No death was observed during the study period.One patient had mild bile leakage,2 patients had cholangititis,and another patient had biliary intestinal anastomotic bleeding.Three patients received resection and reconstruction of the portal vein.Three right hepatic arteries and 1 anterior branch of right hepatic artery were resected in combination with the tumor because of invasion.All the complications were alleviated under conservative treatment.Twelve cases had been followed up for a median time of 16 months (range 3 ～ 24).Two cases had cholangititis intermittently.One case underwent radiotherapy because of local recurrence in 2 years after the surgery.MRCP and relevant enzymes were within the normal range.Conclusion Modified Chen's biliojejunostomy is a simple,effective and safe method,which can be widely used when there are multiple biliary intestinal anastomoses.

OBJECTIVETo explore the prevalence and the different risk factors for asthma in children between urban and rural areas in Fuzhou, Fujian province.

METHODThe epidemiological survey of asthma in 0-14 years old children was conducted from October 2009 to October 2010 between Fuzhou urban and rural areas in Fujian province. The investigation subjects were selected in urban and rural areas by phased stratified random cluster sampling. The 2010 third national epidemiological survey questionnaire of children with asthma was used for screening for possible patients. Diagnosis of asthma was confirmed by physical examination. The children with asthma were designated as the positive cases, while non asthmatic children who were age, gender, ethnic, and living environment matched with asthmatic patients were designated as negative control. Comparison of the prevalence of asthma in children between Fuzhou urban and rural areas was performed. The influencing factors of asthma were analyzed and screened by the regression equation model of two element Logistic regression.

RESULTTotally 12 235 questionnaires of children with asthma and allergic disease screening were issued and 11 738 questionnaire were sent back (6 221 were male and 5 517 were female). The return rate was 95.9% in urban Fuzhou; 648 children were diagnosed as asthma. The prevalence of asthma in male was 6.48% and female children was 4.44% (comparison of the prevalence of gender χ(2) = 23.267, P < 0.001) in urban areas . A total of 6 000 questionnaires of children with asthma and allergic disease screening were sent out and 5 860 were responded (male children 3 228, female children 2 632). The recovery rate was 97.7% in rural Fuzhou; 135 children with asthma was diagnosed. The prevalence of asthma in male was 2.73%and female children and was 1.79%. Adding protein supplement before 6 months (OR = 1.908, 95%CI:1.233-2.959), the use of antibiotics in the treatment of asthma (OR = 14.541, 95%CI:8.920-23.705), furniture materials (non wood) (OR = 2.432, 95%CI:1.563-3.785) were the main risk factors of children with asthma in urban. Adding protein supplement before 6 months(OR = 3.021, 95%CI:1.357-6.711), the use of antibiotics in the treatment of asthma(OR = 14.784, 95%CI:3.842-56.885), the use of coal as fuel (OR = 63.339, 95% CI: 7.993-501.943), domesticated livestock (OR = 13.659, 95% CI:1.342-139.068), the family smoking before and after birth (OR = 6.226, 95%CI:2.674-14.495) and chemical fiber pillow (OR = 3.638, 95%CI:1.241-10.666) were the main risk factors of children with asthma in rural areas.

CONCLUSIONThe prevalence of children with asthma in urban areas was higher than that in rural areas. The prevalence of asthma in male children was higher than in female children. Adding protein food supplement before 6 months, the use of antibiotics and non solid wood furniture material were the main risk factors in children with asthma in urban areas. Adding protein supplement before 6 months, the use of antibiotics, domesticated livestock, the use of coal as fuel and the family smoking before and after birth were the main risk factor of asthma in children in rural areas.

Adolescent ; Anti-Bacterial Agents ; administration & dosage ; adverse effects ; Asthma ; epidemiology ; etiology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Logistic Models ; Male ; Prevalence ; Risk Factors ; Rural Population ; statistics & numerical data ; Sampling Studies ; Smoking ; adverse effects ; Surveys and Questionnaires ; Urban Population ; statistics & numerical data

Objective To explore the treatment of patients with severe traumatic shock first-aid and the preventive measure of the complications caused by a large number of fast blood transfusion during surgery.Methods Retrospective analysis of 87 patients with severe traumatic shock and rapid blood transfusion given to a large number of patients for emergency medical treatment, while the load cycle, bleeding, body temperature, serum potassium, and other indicators were observed, effects and complieations of treatment and effective prevention measures were summarized.Results 87 patients after aggressive surgical treatment,the success rate was 81.61% (71/87) ;surgery circulatory overload caused massive blood transfusion, bleeding tendency,low body temperature,low blood potassium disorders such specific complications.Conclusion Patients with severe traumatic shock should be taken promptly and effective first aid treatment and.the complication caused by massive blood transfusion during surgery,and it could reduce mortality and complications.