Objective To investigate the role of protein phosphorylation in Pseudomonas aeruginosa (P.aeruginosa) strains in response to stress triggered by mouse macrophages.Methods The strong cation exchange-immobilized metal affinity chromatography (SCX-IMAC) was performed to enrich phosphopeptides.The nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) was carried out to identify and analyze phosphoproteome.Results Fourteen phosphopeptides from twelve proteins were identified within thirty-one phosphorylation sites on serine,threonine and tyrosine residues.Fifty percent of these phosphorylated proteins were membrane proteins,indicating that their phosphorylation modification was more critical for bacteria in response to the stress.In terms of biological process of Gene Ontology,these identified proteins were involved in stress response,iron transport,anaerobic respiration,response to hydrogen peroxide and signal transduction by phosphorylation,etc.Conclusion These phosphorylated proteins in P.aeruginosa strains are necessary for signal transduction and their response to harsh environment within the macrophages,such as iron limitation,hypoxia and oxidative stress.This study provides evidence for further investigation on virulence and pathogenesis of P.aeruginosa.

Objective To evaluate super-selective renal arterial embolization(SRAE)in treating severe renal hemorrhage when conservative treatment had failed. Methods SRAE was performed in 111 patients with severe renal hemorrhage who had failed to respond the conservative management.The clinical data,the way of embolization,the medication and the follow-up findings were retrospectively analyzed.Results Excellent results were obtained in all patients after SRAE and no serious complications occurred.The technical successful rate with single session was 95.5%(106/111).Gross hematuria disappeared within 1-4 days after the treatment.Two patients developed shock after renal embolization and had to receive surgery after the shock was controlled.Three patients had a recurrence of hematuria,the blood urine subsided after SRAE was employed again.A follow-up with a mean period of 37.4 months was carried out in 92 patients,and the follow-up checkups showed that the renal function was well preserved in all patients.Conclusion Super-selective renal artery catheterization and embolization is a safe and effective treatment for severe renal hemorrhage,it can maximally preserve the healthy renal parenchyma as well as the renal function.Therefore,this technique should be regarded as the treatment of first choice for patients with severe renal hemorrhage.

Objective To clone and express the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum (Tp) in Escherichia coli,in an effort to develop serological tests with increased specificity for the diagnosis of syphilis.Methods The gene encoding Tp0453 recombinant outer membrane protein fragment was amplified by polymerase chain reaction (PCR),and inserted into expression vector pQE30 after T-A cloning,then confirmed by restriction map.The constructed recombinant plasmid pQE30-Tp0453 was transformed to E.coli M15 for expression induced by isopropyl β-D-1-thiogalactopyranoside.The expressed product was identified by Western blot,and purified by Ni2+-NTA agarose column chromatography.A double antigen sandwich enzymelinked immunosorbent assays (ELISA) was established by using the recombinant Tp0453 protein to test sera from 48 patients with positive Treponema pallidum particle agglutination test (TPPA),and 40 negative sera as control.Results The PCR amplicon of the target gene was about 490 bp.The recombinant plasmid pQE30-Tp0453 was correctly constructed and successfully expressed in E.coli M15.The expressed product,with a relative molecular of about 21 000,existed in a form of inclusion body,accounting for about 18％ of total somatic protein,and reached a purity of more than 95％ after purification.Western blot showed specific reaction of the expressed protein with Tp positive serum.The ELISA tests with the 88 clinical samples yielded a sensitivity of 97.9％ (47/48),and specificity of 100.0 ％ (40/40).The consistency of results between the ELISA test and the TPPA test was 98.9 ％ (87/88).Conclusion The expressed Tp0453 fragment has showed good immunoreactivity with serum from patients with syphilis,providing the foundation of further development of serological diagnostic kit with increased specificity for the diagnosis of TP infection.

Objective To understand the empiric antimicrobial use in patients with pyelonephritis in a hospital,and pro-vide reference for clinical rational antimicrobial use.Methods Data of 620 patients with pyelonephritis admitted to the nephrology department of a hospital between January 2011 and September 2014 were collected,application of antimicrobial agents,coincidence between empiric antimicrobial use and antimicrobial susceptibility testing results in patients with different diseases and different ages were analyzed.Results Before antimicrobial susceptibility testing results were reported,620 pa-tients use 625 times of antimicrobial agents,5 of whom used two kinds of antimicrobial agents at the same time,8 varieties in 15 types of antimicrobial agents were involved,the most frequently used antimicrobial agents were third generation ceph-alosporins,cephamycins,and fluoroquinolones.The overall,partial,and non-coincidence rate between antimicrobial use and antimicrobial susceptibility testing results were 64.32%(n=402),8.32%(n=52),and 27.36%(n=171)respective-ly.The overall coincidence rate in patients with acute pyelonephritis was higher than those with chronic pyelonephritis (77.61% [n=357]vs 58.79%[n=97],P <0.05).The overall coincidence rate in patients <50 years old and ≥50 years old were 68.12%(156/229)and 75.25%(298/396)respectively,there was no significant different between two groups (χ2 =2.93,P =0.09).Conclusion The non-coincidence rate between empiric antimicrobial use and antimicrobial suscepti-bility testing results is high,measures needs to be taken to improve the empiric antimicrobials use.

Objective　To recombine and express a component of urease B subunit transmembrane protein of helicobacter pylori. Methods　A 732 bp gene fragment of urease B subunit of helicobacter pylori was cloned into pET11C and transformed into BL21（DE3）E.coli. The positive clone was induced with IPTG. The expression of target protein was analysed by SDS-PAGE and Western blot. Results　It is successful to construct the recombinant plasmid pET-UreB0.7 containing urease B subunit 0.7 kb gene fragment. A protein （MW≈28 000 u） with immunoreactivity， was expressed by 19.8％ in BL21（DE3）E.coli induced with IPTG. Conclusions　The recombinant component of urease B subunit transmembrane protein may play a role in the research of its biological function and might be used as the vaccine against helicobacter pylori.

Objective: To compare the histopathological changes after neo-adjuvant radiotherapy and to elucidate the mechanism of radiotherapy in rectal cancer. Methods: 80 patients with rectal cancer in pTNM stage Ⅱ or Ⅲ were enrolled between April 2000 and December 2002.They were randomly assigned to surgery alone or preoperative neo-adjuvant radiotherapy.The conventional radiotherapy scheme was followed: 40 Gy in 2.0 Gy fractions.The treatment last 4 weeks and it is usually followed by an interval of 1-2 weeks before the operation.Pathological changes including necrosis of tumor and changes of matrix and vessels were graded. Results: Significant tumor regression(RCRG 1) was seen in 14 cases(35 percent) after radiotherapy,while partially tumor regression(RCRG 2) was seen in 18 cases(45 percent).Significant necrosis was observed in 72.5 percent of cases after preoperative radiotherapy,most foci of adenocarcinoma were replaced by fibrosis in 80 percent of cases,and intimal thickening in most of the vessels were seen in 77.5 percent of cases.The frequency of these pathological changes after radiotherapy was significantly more than control group. Conclusion: Necrosis,fibrosis and thickening of vascular intima in the rectal cancer tissue after radiotherapy is more frequent than those without radiotherapy.It may be the potential reason for increased resection rate and sphincter-saving after radiotherapy.

Objective:To analyze the therapeutical effect of Allicin for colitis mice induced by DSS and its possible mechanisms. Methods:A total of 24 male mice were randomly divided into Control group,DSS group(2. 5% DSS,7 days) and Allicin group [DSS treatment and Allicin,10 mg/(kg·d),7 d]. Disease activity index,inflammatory score,TUNEL and Western bolt were performed to analyze the effect of Allicin on colitis induced by DSS. Results: With DSS group, the Allicin group had lower disease activity score and inflammatory score(P<0. 05). Allicin group had a lower number of intestinal epithelial cell apoptosis than that of DSS group,the difference was statistically significant(P<0. 05). Western bolt analysis shown that Allicin treatment significantly depressed the expression of p-JAK2 and p-STAT3 in intestinal mucosa when compared with these of DSS group ( P<0. 05 ) . Conclusion:Conclusion Allicin has significantly therapeutic effect on mice colitis induced by DSS, the possible mechanisms including anti-inflammatory effects,protected of the intestinal epithelial cells and inhibition of the JAK2/STAT3 signaling pathways.

OBJECTIVETo explore the effect of human endostatin expressed by host cells on the growth of human liver carcinoma in vivo.

METHODSHuman endostain gene was transferred into SMMC7721 cells by retroviral pLncx to build endostatin-transfected cell line. PCR, immunohistochemistry and Western blot analysis were applied to examine the transfection, expression and secretion of endostatin. Endothelial cell proliferation assay was used to determine the biological activity of expressed endostatin. The in vivo and in vitro growth rates of the endostatin-transfected and control SMMC7721 cells were also observed.

RESULTSPCR proved that the genome of endostatin-transfected SMMC7721 cells contained a 550 bp specific fragment of endostatin. The expression and secretion of human endostatin from endostatin-transfected SMMC7721 cells were confirmed by immunohistochemistry and Western blot analysis. Endostatin expressed by host cells could inhibit the proliferation of human umbilical vein endothelial cells by 48% (P < 0.01). In vitro proliferation assay showed that endostatin-transfected SMMC7721 cells had no change in proliferation rate compared to control SMMC7721 cells. In comparison with control group, however, tumor growth rate in vivo from endostatin-transfected SMMC7721 cells was inhibited greatly by 94.5%, 22 days after inoculation into nude mice (P < 0.01).

CONCLUSIONHuman endostatin mediated by retroviral gene transfer can inhibit greatly the growth of human liver carcinoma SMMC7721 in vivo.

Animals ; Carcinoma, Hepatocellular ; therapy ; Collagen ; genetics ; Endostatins ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Mice ; Peptide Fragments ; genetics ; Retroviridae ; genetics ; Transfection

OBJECTIVETo explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma.

METHODSA recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed.

RESULTSThe expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P < 0.01). The endostatin-transfected SMMC-endo cells had similar in vitro growth rates to SMMC-pLncx cells. The in vivo experiment showed that the growth rate of SMMC-endo cells was slowed. Only in 3 out of 5 mice were tumors formed and flank tumors of SMMC-endo cells were 94.5% smaller than those of control cells 22 days after inoculation into nude mice (P < 0.001).

CONCLUSIONSGene transfer of human endostatin mediated by retroviral vector is an effective form of cancer therapy.

Animals ; Cell Division ; Cell Line ; Collagen ; genetics ; Endostatins ; Endothelium, Vascular ; cytology ; Gene Transfer Techniques ; Genetic Therapy ; Mice ; Mice, Nude ; Neoplasms, Experimental ; therapy ; Peptide Fragments ; genetics ; Retroviridae ; genetics ; Transfection

Objective To discuss the experience of medical treatment and rescue of the ‘SHENZHOU' astronauts in the landing place in the past 10 years,and to establish more effective mobile ICU in medical helicopters to ensure ‘SHENJIU' astronauts safety.Methods The data collected from foreign nations and our country was reviewed.Formerly experience in first-aid and rescue astronauts of our team was summerized.The important reasons of accidental injuries of astronaut during aerospace flight were listed.More effective and reasonable prophylactic measurements and clinical treatments of the accidental injuries of astronauts were brought forward.Results we established three effective mobile ICU that could ensure the safety of the astronauts.The carriers of the ICU were helicopters,and damage control surgery can be performed there.Fine armature made us more effective.Conclusions Reasonable and effective prophylactic measurements and clinical treatments were the important aspect of the successful manned aerospace flight.The first-aid system in helicopters could realize the destination of swift response and first-aid.