Bacterial infection has been implicated in premature labor in human. But it is impossible to undergo human study of bacteria-induced preterm delivery. If we carry out animal experiment which simulate human preterm delivery induced by bacteria, studies for mechanism, diagnosis, and treatment of preterm delivery will be progressed rapidly. To elucidate mechanisms and potential intervention strategies in preterm pregnancy loss, we observed bacteria-induced preterm labor and the protecting effect of administration of antibiotics with hysteroscopy-guided intracervical inoculation of Escherichia coli. Sterile saline solution(group I, n=5) or 2x10(7)cfu (colony-forming units) of E. coli bilaterally in the cervix of pregnant New Zealand White rabbits on day 20 or 21(70% of gestation) by hysteroscopy was inoculated and rabbits were assinged to ampicillin-sulbactam therapy beginning at 0hr(group II, n=4), 2 hr(group III, n=4), 4 hr(group IV, n=2), and 16 hr(group V, n=2) after inoculation with E. coli, or to no antibiotic therapy(group VI, n=3). Unasyn(ampicillin-sulbactam) was used and its daily dosage was 100 mg/kg/day. The occurrence of vaginal bleeding or preterm birth was observed every two hours. If one rabbit fetus was found to be delivered, exploratory laparotomy was done. Amniotic fluid culture on each sac, decidual culture on each uterine cavity, and pathologic examinations on each placenta were done. The results of experiments are as follows. In control group(0.2cc sterile saline inoculation only), there was no preterm labor and no bacterial growth in culture. In all three rabbits in group VI, preterm delivery occurred and the culture results were all positive in maternal blood, decidua, and amniotic sacs. Preterm delivery also occurred in group V, but results of maternal blood culture were all negative. Increased trend in the occurrence of preterm delivery was statistically significant in the order(p < 0.05) : group I(0/5), group II(0/4), group III(0/4), group IV(0/2), group V(2/2), and group VI(3/3). Pregnancy outcomes on the basis of the number of living fetus, dead fetus, and macerated fetus, have significant trend in the above order. Amniotic fluid culture results also had significant relationship(p < 0.05) : group I(0.20), group II(20/26), group III(18/30), group IV(10/11), and group VI(7/7). In group V, amniotic fluid fail to be obtained due to severe oligohydramnios. Decidual culture results also had an increased trend; group I(0/32), group II(21/29), group III(20/30), gorup IV(16/16), gorup V(11/11), and group VI(25/25). It is statistically significant(p < 0.05) Incidence of histologic chorioamnionitis was also significantly increased from group I to VI. These results indicate that E. coli inoculation has induced preterm delivery and antibiotic therapy has somewhat prevented preterm birth, amniotic fluid infection, decidual infection, and histologic chorioamnionits. Antibiotic effects were attenuated in cases of delayed antibiotic administration.
Amniotic Fluid ; Animal Experimentation ; Anti-Bacterial Agents* ; Bacteria ; Bacterial Infections ; Cervix Uteri ; Chorioamnionitis ; Decidua ; Diagnosis ; Escherichia coli ; Female ; Fetus ; Humans ; Hysteroscopy ; Incidence ; Laparotomy ; Models, Animal ; Obstetric Labor, Premature ; Oligohydramnios ; Placenta ; Pregnancy ; Pregnancy Outcome ; Premature Birth ; Prognosis* ; Rabbits ; Uterine Hemorrhage

0.05). CONCLUSIONS Levofloxacin is suited to prevent and treat the infection during the perioperation of appendectomy due to acute appendicitis.

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.

In order to realize the sustained education concept in clinical medical English teaching,several measures were taken in the first clinical medical college of Nanjing University of Traditional Chinese Medicine,such as training the teaching staff,using original textbooks and redesigning the curriculum.Particularly the tutorial system was introduced to the education frame.The teaching and research section of clinical medical English explored the new teaching routes for postgraduates in traditional Chinese medicine university.

CD56 Antigen ; metabolism ; Carcinoid Tumor ; diagnostic imaging ; metabolism ; pathology ; surgery ; ultrastructure ; Chromogranin A ; metabolism ; Follow-Up Studies ; Humans ; Kidney Neoplasms ; diagnostic imaging ; metabolism ; pathology ; surgery ; ultrastructure ; Male ; Microscopy, Electron, Transmission ; Middle Aged ; Phosphopyruvate Hydratase ; metabolism ; Synaptophysin ; metabolism ; Tomography, X-Ray Computed ; Vimentin ; metabolism

Objective To evaluate the value of GE′s Q-analysis real-time ultrasonic elastography quantitative analysis software in the diagnosis of breast diseases and explore the best cutoff point of the breast diseases diagnosis.Methods The elastograms of 71 breast lesions(66 patients)were evaluated by using the GE LOGIQ E9 with the Q-analysis software.All the 71 lesions were diagnosed as malignant and benign lesions by surgery or biopsy.The receiver operating characteristic (ROC)curves of the entirety elasticity rate and local elasticity rate was performed.We compared the areas under ROC curves ( AUC) of the two modalities and confirmed the best cutoff point of the breast diseases diagnosis .We also assessed the sensitivity and specificity of the two modalities .Lastly we assessed the ROC curves with Z test to determine whether the difference between the two modalities was significant .Results Among the 71 leisions (66 patients),there were 37 benign lesions and 34 malignant ones.The elastograms of the leisions showed that the elasticity of 37 benign leisions was good ,the hardness value was low and the texture was even .As for the 34 malignant leisions,the elasticity was bad,the hardness value was high and the texture was uneven .The AUC of the local modality was 0.899 ( >0.5).The AUC of the entire modality,was 0.985 ( >0.5).Both modalities were accurate for the diagnosis of breast diseases .When the cutoff point of entirety elasticity rate was set as 3.60,the sensitiveness was 82.4%and the specificity was 86.5%.When the cutoff point of local elasticity rate was set as 2.60,the sensitiveness was 97.1% and the specificity was 91.9%.The difference between the two modalities was significant (Z =2.621,P=0.0088).Conclusions Q-analysis real-time ultrasonic elastography quantitative analysis is able to evaluate the rigidity and texture of tissue and is valuable to distinguish benign breast lesions between malignant breast lesions .The sensitiveness and specificity of local modality are higher than that of entirety modality .

Objective To explore the variance of plasma brain natriuretic peptide (BNP) concentrations in the aged patients after noncardiac surgery and its significance. Methods 101 patients undergoing elective noncardiac surgery were divided into two groups based on the BNP concentrations before surgery: group A: BNP≤100 ng/L，n=61; group B: BNP>100 ng/L，n=40. The BNP concentrations before and after noncardiac surgery and the incidence of cardiac events in both groups were compared. Results There was no significant difference （P>0.05） of BNP concentrations before and after noncardiac surgery in group A, which were (58.2±28.7) ng/L and (53.7±25.9) ng/L respectively, but was significant difference （P<0.05） in group B, which were (147.3±72.1) ng/L and (341.5±92.4) ng/L respectively. There was significant difference （P<0.05） between group A, in which no patient happened cardiac event, and group B, in which 14 patients happened. Conclusion The plasma BNP concentration would be increased significantly in the aged patients with a BNP concentration>100 ng/L before surgery, which may cause more cardiac events.