Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire pimQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expres-sion products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneoualy immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminloglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQ mutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nueleotide and putative amino acid sequences of the cloned phop and phoQ genes were 98.7%-99.6% and 98.7%-100% , and 98.4%-99.8% and 99.1%-100%, respec-tively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coil BL21 DE3 system, and their rabbit antisera with 1 : 4 and 1 : 8 double immunodiffusion titers were also obtained. The deletion of phoQ genes and absence of the products in the two phoQ mutants were confirmed by PCR, sequencing and West-ern blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512-1/2048 as those of their wild strains. Conclusion PboQ/PhoP is a sequence conserved two component sig-naling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.

Liver tissues around hydatid from 8 patients with cystic echinococcosis were observed by transmission electron microscopy,normal hepatic tissues of 6 cases were used as control.The results demonstrated predominant atrophy and necrosis of hepatocytes.These changes seem to be the major hepatic lesion in cystic echinococcosis.

Objective To evaluate the dosimetry of high-energy electron beams by using GafChromic EBT film. Methods The percent depth doses of electron beams of 4 MeV,6 MeV,8 MeV,10MeV, 12 MeV and 15 MeV were measured with EBT. The results were then compared with the measurements with diode detector in RFA and parallel plate chamber in water tank. Results The percent depth dose curves using EBT film had a good agreement with those using the other two detectors. Furthermore, no differences were found between up-right and tilt setup methods. When film upper edge is higher than water surface, a sharp drop of measurement results using EBT film was observed in comparison with those from diode detector in build-up region. Conclusions EBT film can be applied to measure percent depth doses of high-energy electron beams. During the EBT film measurements, the film should be tilted at the angle of 5degrees to the central axis of the field. When choosing up-right setup method, the edge of the film should be parallel to the water surface.

Objective:To inspect the dosimetry characteristic of a 2-D ionization chamber array and its response to oblique incident electron beam.And to discuss feasibility to using it to validate the electron arc therapy plan.Materials and methods:(1)The 2-D ionization chamber array was calibrated referring to a themble chamber,comparing the response to electron beams with incidence angle ranged from-50°to 50°.(2)Phantom test plans of 6 and 10MeV electron beam arc therapy were designed,with the beam rotation arcs of 30°,60°and 90° respectively,symmetric to the Central axis of 0°gantry angle.Dose distribution of each plan was verifled with the 2-D array.Results:(1)Measurement differences between the central detector of the array and the thimble chamber were less than 2%,under the planned oblique inddent 6/10MeV electron beam.(2)For the all test plans,dose error was less than 3%at dmax in the symmetric axis of the electron beam arcs.Off-axis dose error was less than 2%within the central 70%area of the profile in non-rotating direction,and smaller than 1.5%in the rotational direction.The measured dose distribution had good agreement with the TPS calculation in isodose curves between 100%and 20%.The gammapass rates(△D=5%,△d=5cm)were 99.98%、99.89%、99.74%、98.64%、99.16%and 99.44%respectively for 6 and 10 MeV plans with electron beam arcs of 30°,60°and 90°.Conclusion:it is practicable using the tested 2-D ionization chamber array to verify and validate the electron beam arc therapy plan.

Objective To determine,in our experience,the prevalence and presence of risk factors for metabolic syndrome (MS) in heart transplant recipients.Methods We studied 135 patients who underwent heart transplantation in our hospital from September 2000 to December 2011,According to the diagnostic criteria of MS,they were divided into two groups.All variables that could be related to the development of MS during the follow-up period were analyzed.The prevalence and presence of risk factors for MS in our experience were determined.Student t-test,Wilcoxon rank sum test and x2 test were used for univariate statistical analysis and logistic regression for multivariate analysis.Results (1) Of the 135 patients,63 developed MS (46.67％) during a mean follow-up period of 47.3 months.The multivariate analysis identified the follows as predictive factors for the development of MS:age (OR=1.09,95％ CI 1.01-1.22,P＜0.05),body mass index (BMI) (OR=1.65,95％ CI 1.16-1.87,P＜0.05),history of DM (OR =3.27,95％ CI 1.12-34.21,P＜0.05).Conclusion In our population,the prevalence of MS after heart transplant is 46.67％ during a mean follow-up period of 47.3 months.Age,pre-operative overweight especially BMI,and history of DM were significant and independent risk factors for the development of MS during the follow-up period.For these patients,dose of immunosuppressant and diet control are important.

Objective To investigate the function of phosphatidylinositol phospholipase C encoded by LB361 gene of L.interrogans ( L-PI-PLC) and its mechanism in inducing macrophage apoptosis .Meth-ods The PI-PLC domains in the sequence of LB 361 gene of L.interrogans serovar Lai strain were analyzed by bioinformatics method .Prokaryotic expression system was established to express the recombinant L -PI-PLC ( rL-PI -PLC).The enzymatic activity of rL-PI-PLC in hydrolyzing phosphatidylinositol -4,5-bisphos -phate (PIP2) substrate into inositol-1,4,5-trisphosphate (IP3) was determined by IP3 fluorescence polariza-tion assay.LB361gene expressions at mRNA and protein levels as well as the secretion of LB 361gene prod -ucts were detected by real-time fluorescent quantitative RT -PCR and Western blot assay after infection of hu-man THP-1 macrophages with L.interrogans serovar Lai strain.A LB361 gene-transfected THP -1cell line was generated for evaluation of the mechanism of LB 361 gene products in elevating intracellular free Ca 2+( [Ca 2+] i) concentration and inducing the apoptosis of transfected THP -1 cells with the use of laser confocal microscopy and flow cytometry.Re sul ts The rL-PI-PLC hydrolyzed PIP2 into IP3 with a Km of 199 μmol/L and a Kcat of 8.566×10-5 S-1 .The expressions of LB361gene at mRNA and protein levels were both signifi -cantly up-regulated after infection of THP-1 cells with L.interrogans serovar Lai strain .Moreover , the exter-nal secretion of L-PI-PLC was also found during infection .The concentrations of IP 3 and [ Ca2+] i in the LB361 gene-transfected THP-1 cells were significantly increased compared to those in the non-transfected THP-1 cells, resulting in a high [Ca2+]i-dependent apoptosis of partial THP-1 cells.Conclusion PI-PLC is encoded by the LB361 gene of L.interrogans, which could induce the apoptosis of macrophages through el-evating [ Ca2+] i concentration during infection of microphages with L.interrogans.

Objective To study the indications for extubation of percutaneous transhepatic gallbladder drainage(PTGD).Methods Between January 2009 and December 2013,PTGD were performed on 104 patients with acute cholecystitis.The clinical data were analyzed retrospectively.Results PTGD were performed on all 104 patients successfully.In all the patients,after drainage the abdominal pain was relieved within 0.5 to 8 hours and mean time was(2.3 ± 1.9) h.The temperature decreased to normal in 1 days after drainage.The time of drainage was from 7 to 49 days,(22.4 ± 10.3) days on average.Thirteen patients'tubes were accidentally off during 3 to 12 days,5 patients were performed PTGD again,and 8 patients were given anti-infection,fluid infusion and other symptomatic treatments and all these patients were back to normal in time.Re-intubation rate was 4.8％.In 78 patients laparoscopic cholecystectomy (LC) were performed 1 to 2 months after PTGD.Operation time was between 35 to 95 min and mean time was(52.7 ± 15.0) min.There were 6 patients done open operation and the conservation rate was 7.7％.A mount of blood during LC was 30 to 150 mL,(61.7 ± 31.0) mL on average.No operative deaths occurred during therapeutic session.Conclusions Patients who formed PTGD obey the indications can extubate in time and reduce retrograde infection and skin irritation,also shorten the time during hospital's,which will have a positive impact about LC.

This study retrospectively reviewed 9 cases of complicated hepatic cystic hydatidosis with intrabiliary rupture who were surgically treated with pericystectomy in combination with Roux-en-Y hepaticojejunostomy in our hospital from 2004 to 2010. The clinical features, results of laboratory tests, B-mode ultrasonography and CT, post-operative recovery, days of hospital stay after the operation and post-operative complications were statistically analyzed and the patients were followed up. The subjects in our series included 7 males and 2 females, whose average age was 50.78±7.58 years. Before operation, 9 patients suffered from pain of the right upper quadrant and jaundice, which, in 4 cases (44.45%), were accompanied with fever and chills. Preoperative B-mode ultrosonography and CT showed that all the 9 patients had single hydatid cyst, with their diameter being 9.33±1.58 cm on average. The lesions involved segments V, VI in 6 cases, and segment IV in 3 cases. By WHO classification, 7 cases were classified as CE3 and 2 cases as CE4. They all had choledochectasia. The subjects underwent the surgery uneventfully. Intraoperatively, 2-4 biliary fistula orifices were found, with the average of the orifice being (0.79±0.20) cm. After the operation, one patient developed incision infection, one had pulmonary infection and one suffered from reflux cholangitis. No anastomotic leaks or peri-operative deaths took place and follow-up revealed no recurrence and implantative metastasis. We are led to conclude that pericystectomy in combination with Roux-en-Y hepaticojejunostomy can achieve satisfactory results for the treatment of complicated hepatic cystic hydatidosis with intrabiliary rupture.

Objective To explore the value of laparoscopy in the diagnosis and treatment of recurrent inguinal hernia in children.Methods The clinical data of 67 cases receiving laparoscopic treatment for recurrent inguinal hernia in children at the Department of General Surgery,Wuhan Children's Hospital,Tongji Medical College,Huazhong University of Science & Technology were retrospectively analyzed,including the type of hernia recurrence,operation time,intraoperative and postoperative complications,such as short-term hydrocele formation and testicular atrophy and so on.Results In 67 cases,there were 65 males and 2 females.Open repair surgery and laparoscopic surgery were respectively performed in 48 cases and 19 cases for the first operation and the hernia defects were found after operation.Contralateral patent vaginal process deformities were found in 11 sides in the reoperation.Among 67 cases of recurrent inguinal hernia,incomplete ligation of hernia sac was found in 21 cases,low level ligation of hernia sac in 17 cases,no ligation of hernia sac in 14 cases,omissive direct inguinal hernia in 4 cases,loose ligation of hernia sac in 4 cases,overlarge inner ring in 4 cases,weak abdominal muscles in 2 cases,and increased abdominal pressure (long-term constipation,asthma) in 1 case.All the patients were treated by laparoscopic hernia repair and no serious complications occurred during the operation.The average time of unilateral operation was (15.0±2.2) minutes (13-25 minutes),and bilateral operation was (27.0±4.3) minutes (18-41 minutes).All patients were discharged on the second day.Two patients showed short-term postoperative hydrocele and were cured with conservative treatment.During the follow-up time of (23±2) months (9-39 months),no recurrence or testicular atrophy was found.Conclusions Laparoscopy can confirm the type of recurrent inguinal hernia and contribute to reduce postoperative recurrence.Laparoscopic management of recurrent inguinal hernia in children is safe and feasible,which is expected to replace open hernia repair.