In present study, standard method and standard operation practice for measuring the activities of influenza neuraminidase and its inhibitors have been established. The accuracy and stability of the method has been evaluated. Standard operation is as following: 10 microL sample, 30 microL neuraminidase and 60 microL substrate are added to one well of a 96-well plate, and then incubated at 37 degrees C for 1 h. The reaction was stopped with NaOH before fluorescence intensity determination. One unit of neuraminidase is defined as the amount of enzyme that produces 1 nmol 4-MU in 1 h under above conditions. The inhibition accuracy is indicated by an uncertainty measurement of 6.51 x 10(-12), and its stability was reaffirmed by determination of oseltamivir acid. In this study, systematic assessment of neuraminidase inhibitory assay not only provided theoretical basis of its application in drug discovery, but also made preliminary attempt to use uncertainty measurement as a parameter in biological measurement.

Objective:To observe the effect of old RBCs transfusion on cognitive function in rats and the improvement effect of mi -nocycline.Methods: Male SD rats at the age of 6 months were randomly divided into 4 groups.The RBCs were obtained from male rats by centrifuging the total blood and stored at 4℃.The rats of fresh RBCs group (group F) were transfused with the RBCs stored for 1 day.The rats of old RBCs group (group O) were transfused with the RBCs stored for 7 days.The rats of treatment group (group T) received 40 mg· kg-1 minocycline with intraperitoneal injection before the transfusion .The rats of the control group ( group C) were transfused with the normal saline .The brain levels of IL-1βand IL-6 were determined with Quantikine ELISA kits in 24 hours after the blood transfusion (n=6).The rats were subjected to Barnes maze tests after 1 week of the blood transfusion (n=10).Results:The brain levels of IL-1βand IL-6 in group O were higher than those in group C and F (P<0.05), which were lower in group T than those in group O(P<0.05).The rats of group O spent longer time finding the target box than those of group C and F in the Barnes maze (P<0.05), and the time was shorter in group T than that in group O (P<0.05).Conclusion: Old RBCs transfusion plays a role in neuro-inflammation and induces cognitive dysfunction in rats , which may be improved by minocycline .

Objective To analyze the cause of platelet aggregation in blood specimens ,so as to provide basis for reducing platelet aggregation ,and avoiding false positive of platelet count ,false report ,misdiagnosis and mistreatment .Methods The blood speci-mens which platelet was below 80 × 109 /L ,below 125 × 109 /L with histogram hinted platelet aggregation ,were smeared ,stained with Wright-Giemsa ,and observed by microscope for platelet morphological changes .The data between each groups were calculated and analyzed by statistical software SPSS version 18 .0 .Results A total of 184 cases of ethylenediaminetetraacetic acid dependent pseudothrombocytopenia(EDTA-PTCP) were found ,accounted for 0 .444 ‰ totally ,including 0 .244 ‰ of out-patients (101 cases) , 0 .159 ‰ of hospitalized patients (66 cases) ,and 0 .041 ‰ of health examination personnel (17 cases) .3 cases of multi-dependent pseudothrombocytopenia and 25 cases of pseudo platelet aggregation were found ,and accounted for 0 .007 ‰ and 0 .060 ‰ respec-tively .Conclusion The discovery of platelet aggregation which caused mainly by EDTA-PTCP ,still relies on microscopy ,and pseu-do platelet aggregation comes mainly from sampling ,so it needs to strengthen the skills training .

Objective To investigate the effects of pentobarbital sodium on compound muscle action potentials (CMAPs) in rats.Methods Ten adult Sprague-Dawley rats (5 males,5 females),aged 8 weeks,weighing 240-260 g,were anesthetized with intraperitoneal 1％ pentobarbital sodium 40 mg/kg.The sciatic nerve was stimulated (intensity 0.50,0.55 and 0.60 V,wave length 0.05 ms,frequency 10 Hz) starting from 8 min after administration.Each intensity was repeated three times at 1 s interval.The stimulation mentioned above was repeated every 5 min.CMAPs from the gastrocnemius muscle were recorded starting from 8 min after administration (T1) and then were recorded every 5 min for 9 times (T2-10),Results The peak value of CMAP was significantly decreased at T3-5 when the intensity was 0.50,0.55 and 0.60 V,and CMAP latency was significantly prolonged at T3-6 when the intensity was 0.50 V,and at T4,5 when the intensity was 0.55 and 0.60 V as compared with those at T1 ( P ＜ 0.05 or 0.01 ).Conclusion Pentobarbital sodium can inhibit CMAPs in rats.

Objective To investigate the long-term excessive drinking semen nitric oxide (NO) content in their sperm quality and spermatogenic cell apoptosis and infertility. Methods Nitrate reductase method was used to specific reduce the NO metabolites nitrate (NO3-) to nitrite (NO2-), which was used on behalf of the total NO level. Terminal deoxynucleotidyl transferase (TdT) and mediated nick end labeling (TUNEL) method and binocular optical microscope were used to detect and observe the rate of apoptosis of spermatogenic cells and the morphological structure. The SQA-V sperm quality automatic analyzer was used to measure sperm quality. Results In not drinking semen fertility group, NO content was (54.81±11.45)μmol / L, the rate of spermatogenic cell apoptosis was (4.52±1.23)%, sperm motility was (80.24±0.17)%, energy a+b (78.32±0.12)%, deformity rate (5.30±0.13)%, and long-term excessive alcohol infertility C group was [(128.83±22.73)μmol/L,(17.34±2.53)%,(51.18±0.58)%,(21.45±0.26)%,(21.12±3.24)%] respectively. Compared to a very significant difference (t＝10.04,17.38,6.69,15.59,17.02,P＜0.01) . In long-term excessive drinking group, the levels of NO and spermatogenesis cell apoptosis rate was significantly positive correlated (r＝0.93,P＜0.01).Apoptosis of spermatogenic cell nucleus chromatin was condensed in the formation of crescent-shaped perinuclear, nuclear was cleavaged to form apoptotic bodies. Conclusions In the long-term excessive drinking semen, NO content and spermatogenic cell apoptosis rate was increased, the sperm had poor quality. The results showed that the long-term excessive drinking can cause germ cell apoptosis in male infertility and promote the body to overproduce NO, whichmay be one of the reasons for male infertility.

Objective To investigate the immunological properties of Rv1009 domain. Methods BALB/c mice were immunized with Rv1009 domain three times at 2-week interval. ELISA was used to detect the antiRv1009 domain antibody titer in the sera of immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. Levels of secreted IFN-γ, IL-10 and IL-12 upon specific antigen stimulation were detected by ELISA. The BALB/c mice immunized with Rv1009 domain were intravenously infected with MTB H37Rv. Four weeks after the final injection, the number of CFU in spleens was determined. Results The titer of the specific antibody in sera of the immunized BALB/c mice was 1:12 800. The SI of Rv1009 domain immunized group (2. 40±0. 18) was significantly higher than that of saline immunized group (0.90±0.21). The IFN-γ,IL-10 and IL-12 levels in culture supematant of spleen lymphecytes from the fusion proteins immunized mice was (1 432±30) ng/L, (503±11) ng/L and (311±11) ng/L respectively, significant different from that of saline immunized group[(256±20) ng/L, (76±6) ng/L and(56±8) ng/L,P<0.01]. Four weeks after the final injection,compared with normal saline immunized mice (6.64±0.13), dramatic reduction in MTB replication was observed in the spleen (4.86±0.14) from BALB/c mice immunized with fusion proteins following a subsequent MTB H37Rv challenge, but the protection efficacy of mice immunized with Rv1009 domain was not as good as that of BCG vaccination group (3.81±0.16). Conclusion Rv1009 domain can be used as a candidate for the new TB vaccine.

Objective To analyze the risk factors of acute kidney injury after orthotropic liver transplantation for adult benign end-stage liver disease.Methods A retrospective analysis was conducted in 30 recipients (18 males,12 females,aged 23-68 years,ASA grade Ⅲ or Ⅳ) who underwent orthotropic liver transplantation for benign end-stage liver disease at Tongji Hospital from May,2014 to December,2014.Both demographic characteristics and perioperative parameters were collected,including general condition,surgery and anesthesia factors and intraoperative salvage autotransfusion or not.Perioperative laboratory findings related to renal function including urine volume,serum creatinine (Scr) and urea nitrogen (BUN) were collected,too.All variables tested in the univariate analysis with a P<0.10 were included in a multiple logistic regression analysis.Results There were less intraoperative salvage autotransfusion,more platelet transfusion and a higher using rate of vasopressors in the AKI group after surgery than those did not.Patients who received intraoperative salvage autotransfusion had 0.058 time odds (95%CI 0.005-0.649) of AKI than those did not;patients who required platelet transfusion had 10.706 times higher odds (95%CI 1.212-94.963) of AKI than those did not.Conclusion It is likely that intraoperative salvage autotransfusion was able to decrease the morbidity of AKI,while platelet transfusion and vasopressor administration to maintain blood pressure could increase the possibility of AKI.

Objective To investigate the mechanism of sleeve gastrectomy combined with modified jejunoileal bypass (SG/MJIB) in improving the blood glucose of Goto-kakizaki (GK) rats.Methods According to the random meter method,the 28 GK rats were divided into 4 groups:sleeve gastrectomy with modified jejunoileal bypass (SG/MJIB),sleeve gastrectomy (SG),modified jejunoileal bypass (MJIB) and sham operation (SHAM).The changes of weight,food intake,fasting blood glucose,oral glucose tolerance test (OGTT),insulin tolerance test (ITT),plasma insulin and gastrointestinal hormonal (ghrelin and GLP-1) were monitored before and 16 weeks after surgery respectively.Results After surgery,the fasting blood glucose level in SG/MJIB group was obviously lower than that in SG,MJIB and SHAM groups (P＜0.05).From the 2nd week after surgery,OGTT of the SG/MJIB group was obviously improved compared to SG,MJIB,and SHAM groups (P＜0.02).In our post-op erative study,the insulin levels in SG/MJIB group were lower than those in SG,and SHAM groups (P＜0.05).Compared to MJIB and SHAM groups,ghrelin levels of SG/MJIB group were significantly decreased (P＜0.001),while GLP-1 levels of SG/MJIB group were higher than those of SG and SHAM groups (P＜0.01).Conclusions SG/ MJIB can improve insulin sensitivity and insulin secretion,and this effect is independent of the body weight and food intake.This study further validates that the gastrointestinal hormones play an important role in the pathogenesis and treatment mechanisms of type 2 diabetes mellitus.

Objective To investigate the relationship between clue cell in semen and male infertility. Method Semen specimens from 957 patients were examined with microscope. Gram staining was performed when clue cell were found to be present in semen. At the same time, general characteristics of the sperm were analyzed. Results There were clue cells in 23. 5% (225/957) of the total specimens, in which, 95. 1% clue cells were gram staining positive. The concentration and vitality of living sperm in semen presented with clue cell were significantly lower, but the abnormal sperm and pH value were much higher than that in normal control (P<0.01). In addition, 28 sexual partners of 30 infertility patients were identified as having bacterial vaginosis. Conclusion Gardnerella vaginalis and short coccohacillus were mostly transmitted from infected sexual partner. It could cause squamous epithelial cell forming clue cell, and lead to infertility by chan-ging pH which mainly affect reproduction of the sperm. This study suggested that clue cell has a unique vale for the diagnosis of infertility causing by Gardnerella vaginalis and short coccohacillus.

Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to in-vestigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro-EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. Coli DHSa and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. Luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. Tuberculosis H37Ra which were in non-culturable' condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 x 103 and 12 x 103, the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. Luteus Rpf and Rpf domain from the E. Coli showed activity of stimulating the resuscitation of M. Luteus and M. Tuberculosis H37Ra in non-cuhurable' condition which could be inhibited by monoclonal antibodies of M. Luteus Rpf domain remarkably. Conclusion It was dem-onstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. Luteus Rpf domain protein is fully active as M. Lateus full-length Rpf.