Objective To construct 2 recombinant plasmids carrying cassette chromosome recombinase C(ccrC)and ccrAB respectively and introduce the plasmids into methicillin-resistant Staphlococcus aureus(MRSA)strain 81/0432,and to observe the precise excision of Staphylococcal cassette chromosome mec(SCCmec)island from bacterial chromosome.Methods ccrC and ccrAB genes were amplified with chromosomal DNAs isolated from MRSA strains 81/0342 and N31S as PCR templates.We constructed recombinant plasmids pSR5C and pSR2AB by cloning ccrC and ccrAB genes into temperature-sensitive plasmid pYI3,after introducing them into MRSA strains 81/0432 and N315 by electroporation.PCR was performed to identify SCCmec excision from the bacterial chromosome.The transformants were serial passaged for 10 days,and then the drug resistance of these rransformants was detected by replica experiment.Results The fragment length of ccrC gene was 1.9 kb,smaller than the fragment length of ccrAB from N315.The recombinant plasmids of pSR5C and pSR2AB were successfully constructed.After these 2 recombinant plasmids were introduced into MRSA strain 81/0342,type-V SCCmec island was excised from the chromosome and formed a closed circular structure in the bacteria.However,type-Ⅱ SCCmec island could be excised only in N31S strain after the expression of pSR2AB.Replica experiment verified that transformed strains of 0342(pSR2AB),0342(pSR5C),and N315 (pSR2AB)were mostly susceptible to ceftizoxim or tobramycin after the excision of SCCmec island.Conclusion cciC could serve as a recombinase as ccrAB,which could mediate precise excision of SCCmec island from the chromosome of type-V MRSA strain.This study shows that type-V SCCmec island is widely disseminated between Staphylococcus aweus strains in community setting.

Objective The study aimed to observe the influence of Puritybile capsules combined with different doses of Compound polyethylene glycol (PEG) in bowel preparation.Methods 120 patients were set into three groups (group A,B and C) randomly (40 patients in each).Group A take the routine Compound polyethylene glycol treatment orally.On the basis of the same routine Compound polyethylene glycol treat-ment,group B added regular dose of Puritybile capsules.Group C took half dose of Compound polyethylene glycol and regular dose of Puritybile capsules.The cleaning effect and adverse reaction were compared among three groups.Results According to Ottawa Scale,the cleaning effects as well as the existing of air bubbles showed no significant difference among three groups.Among which group C was 30％lower than that of group A,10％ lower than that of group B.There was no statistical differences in adverse events.Conclusions Half dose of Compound polyethylene glycol and regular dose of Puritybile capsules can guarantee the ef-fect of bowel cleaning and lower medical expense of patients.

Anti-glutamic acid decarboxylase antibody-associated encephalitis is a kind of autoimmune encephalitis mediated by anti-glutamic acid decarboxylase antibody, which belongs to anti-neuronal intracellular synaptic protein antibody-associated encephalitis. Clinical manifestations include stiff-person syndrome, cerebellar ataxia, limbic encephalitis, seizures, etc., often associated with a variety of autoimmune diseases, rarely associated with tumors. Detection of anti-glutamic acid decarboxylase antibody is crucial for clinical diagnosis. Immunotherapy helps to relieve symptoms and improve prognosis. The incidence of this disease is low, and there are few reports at home and abroad. This paper intends to review the research on this encephalitis, hoping to improve the clinicians′ understanding and the level of diagnosis and treatment of the disease.

Objective Scutellarin (SCU), a Chinese traditional medicine, has a protective effect against ischemia-reperfusion (IR) induced myocardial injury, but it is not yet clear whether SCU acts against vascular endothelial IR injury via extracellular signal-regulated kinase 1/2 (ERK1/2).The aim of this study was to explore the effect of SCU on hypoxia-reoxygenation (HR)-induced injury to human cardiac microvascular endothelial cells (HCMECs) and its influence on the ERK1/2 signaling pathway.Methods HCMECs were subjected to normal culture and divided into a normal control, a DMSO, an SCU 1 μmol/L, and an SCU 10 μmol/L group.The model of HR injury was established by exposing the HCMECs to 12-h hypoxia and 12-h reoxygenation after treated with DMSO or SCU at 1 and 10 μmol/L for 2 hours.Then, the survival rate of the HCMECs was detected by MTT and trypan blue staining, the concentration of malondialdehyde (MDA) in the cells measured, and the expressions of the p-ERK1/2, ERK2 and GAPDH proteins determined by Western blot.Results SCU at 1 and 10 μmol/L significantly increased the survival rate of the normally cultured HCMECs ([110.40±2.34] and [122.00±1.25] ％) as compared with that of the normal control (100％) (P<0.05), while HR injury markedly decreased the vitality of the HCMECs ([68.00±4.06] ％) in comparison with that of the blank control (100％) (P<0.05).The survival rate of the HCMECs was remarkably higher in the HR+SCU 1 μmol/L and HR+SCU 10 μmol/L groups than in the HR model group ([90.53±3.67] and [92.04±2.32] ％) (P<0.05), and so was their vitality in the SCU 10 μmol/L group than in the normal control ([96.78±2.01] vs [90.06±1.85] ％, P<0.01), while their survival rate was significantly lower in the HR model than in the blank control ([73.72±4.91] vs [91.83±2.34] ％, P<0.01) and remarkably higher in the SCU 10 μmol/L ([87.59±2.64] ％) than in the HR model group (P<0.05).The MDA concentration in the HCMECs was markedly increased in the HR model and HR+DMSO groups as compared with the blank control (P<0.01), but decreased in the HR+SCU 1 μmol/L and HR+SCU 10 μmol/L groups in comparison with the HR model group (P<0.05).The expression of the p-ERK1/2 protein was significantly down-regulated in the HR model group as compared with the blank control (P<0.01), but up-regulated in the HR+SCU 10 μmol/L group in comparison with the HR model (P<0.01).Conclusion HR injury reduces the vitality of HCMECs, increases the MDA concentration, and down-regulates the expression of the p-ERK1/2 protein in HCMECs, while SCU acts against ischemia-reperfusion injury to HCMECs by increasing ERK phosphorylation.

Objective To obtain highly purified botulinum neurotoxin A light chain(BoNT-ALC) protein in E.coli by genetic engineering and multi-step purifications, and identify its metalloproteases activity.Methods The full-length of BoNT-ALC was cloned from BoNT A by PCR and inserted into plasmid pET-22b.Then pET-22b-ALC was transformed into E.coli BL21( DE3) strains and induced by IPTG.The protein was purified by Ni-NTA sepharose,anion exchange column and gel filtration.The enzymatic activity of the protein was identified by SNAP-25.Results and Conclusion A highly purified and homogeneous protein is obtained, which shows good enzymatic activity.

Objective:To investigate the pharmacokinetics of mycophenolic acid( MPA)and its metabolites in different stages af-ter the administration of enteric-coated mycophenolate sodium( EC-MPS)tablets in Chinese liver transplant recipients. Methods:The blood samples of 24 patients were collected in 0-12h of the 1st and 3rd week after the administration of EC-MPS. The concentrations of MPA,AcMPAG and MPAG in plasma were measured by LC-MS/MS developed in our lab. The pharmacokinetic parameters of MPA and its metabolites were estimated by non-compartmental method. Results:After 1-and 3-week therapy with EC-MPS,Cmax ,AUC0-12 and t1/2 was(18. 1 ± 8. 75)and(20. 7 ± 16. 0)μg ml-1 ,(42. 7 ± 17. 5)and(47. 1 ± 23. 9)μg·h·ml-1 ,(3. 33 ± 2. 81)and (3.30 ±1.89)h for MPA;(2.50 ±1.86)and(1.78 ±1.72)μg·ml-1,(14.5 ±11.7)and(6.97 ±6.57)μg·h·ml-1, (4. 48 ± 2. 53)and(3. 76 ± 1. 8)h for AcMPAG;(171. 6 ± 135. 4)and(152. 2 ± 115. 9)μg·ml-1 ,(1299 ± 1 204)and(1 051 ± 561)μg·h·ml-1 ,(8. 73 ± 4. 25)and(7. 75 ± 2. 87)h for MPAG,respectively. There was no significant difference in the PK parameters of MPA after the 1-and 3-week therapy. The Cmax ,Tmax and t1/2 of MPA in the patients received EC-MPS were significantly higher than those in the patients received MMF(P<0. 05). Cmax and AUC0-12 of MPAG in the patients received EC-MPS were signifi-cantly higher than those in the patients received MMF after the 3-week therapy(P<0. 05). Conclusion:There is no significant accu-mulation of MPA after the therapy with EC-MPS at different stages. The absorption of MPA is delayed after the therapy with EC-MPS compared with that with MMF. There is no difference in MPA exposure between EC-MPS and MMF in Chinese liver transplant patients.

A high-speed counter-current chromatography (HSCCC) method was successfully developed for the preparative separation and purification of deoxyschizandrin from Schisandrae Sphenantherae Fructus in one step. The purity of deoxyschizandrin was 98.5%, and the structure was identified by MS, UV and NMR. This method was simple, fast, convenient and appropriate to prepare pure compound as reference substances for related research on Schisandrae Sphenantherae Fructus.

Objective To study the effect of mitogen-activated protein kinas/extracellular signalregulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA).Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic synovectomy of RA patients with active disease activity [disease activity score(DAS)28 ≥3.2].The RA-FLS were primarily cultured by the explanted culture,and then were treated with Shh agonist purmorphamine,inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126,respectively.Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2),which was the critical protein of MAPK/ERK signaling.The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8),and the cell proliferation rate was detected using a flow cytometry.Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis.Results Compared with the control group,purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L,and the peak activations of p-ERK1/2 took place at 15 min (P＜0.01).Cyclopamine and U0126 decreased the expression ofp-ERK1/2 protein (P＜0.01).After the RA-FLS treated with purmorphmine(1 μmol/L)for 48 hours,the cell proliferation activity was (114±4)％ and the percentage of S phase cells was (8.39±0.60)％,which was significantly higher than those of the control group (100±0)％ (P＜0.01) and (3.29±0.69)％ (P＜0.01).After treated with cyclopamine (10 μmol/L) for 48 hours,the cell proliferation activity of RA-FLS was (89±1)％ (P＜0.05) and the percentage of S phase cells was (1.53±0.22)％ (P＜0.05).When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L),the cell proliferative activity was (89±2)％ (P＜0.05) and the percentage of S phase cells was(1.07±0.25)％(P＜ 0.05).Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling,which in turn contributes to hyperplasia of synovium and ultimately leading to RA.

Objective The optimal access for natural orifice transluminal endoscopic surgery is still uncertain .This study was designed to compare the practicability and maneuverability of transgastric ,transunmbilical ,and transrectal approach in abdominal surgery in a canine model .Methods Three dogs were used in this research .Three approach :trangastric ,transunmbilical and tran-srectal approach were carried out for abdominal exploration ,liver biopsy ,bladder biopsy and an attempted cholecystectomy .The ma-neuverability ,endoscopic image ,performer′s perception ,and spatial orientation were evaluated .Results The maneuverability of trangastric ,and transrectal approach NOTES were better than transunmbilical NOTES .Abdominal exploration ,live biopsy ,and bladder biopsy were completed successfully .The cholecystectomy was failed because of poor exposure and difficulty of separating the around tissure .Conclusion The optimal approach for upper abdomen NOTES is transrectal route .For lower abdomen NOTES , the trangastric approach is superior to other accesses .Further study is needed to develop more flexible and precise equipment for NOTES and to evaluate more feasible access approach .

Objective:To preliminary study the effects of long noncoding Ribonuclei Acid (RNA) small nucleolar RNA host gene 1 (SNHG1) on proliferation of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and the possible mechanism.Methods:The FLS from RA and trauma group were primarily cultured by the explant culture, and the expression of SNHG1 were detected by quantitative polymerase chain reaction (qPCR). Transfection of siRNA was used to interfere the expression of SNHG1. Cell viability was measured using CCK-8 assay and cell cycle distribution was detected by flow cytometry. And the protein level of cyclinD1 was detected by Western blotting. Independent sample t test was used for the comparison between two groups, and the one-way analysis of variance (ANOVA) analysis was used to compare the samples of multiple groups. Results:Compared with FLS from trauma group, SNHG1 expression in RA-FLS was up-regulated [(2.13 ±0.55) vs (1.00 ±0.01)] ( t=-5.87, P=0.004). In RA-FLS, after silencing the expression of SNHG1, the cell viability of SNHG1-siRNA treatment group was down-regulated [(0.930 ±0.033) vs (0.759 ±0.027)]( t=6.879, P=0.002), the proportion of G 2/M+S cells was down-regulated [(28.2 ±1.5)% vs (9.7 ±2.6)%]( t=10.715, P<0.01), and thelevelof cyclinD1 protein was down-regulated ( t=6.168, P=0.004) compared with the negative control group. Conclusion:The SNHG1 is abnormally expressed in RA-FLS, and SNHG1 may participate in the regulation of FLS proliferation by affecting the expression of cyclinD1 protein, thereby contributing to synovial hyperplasia.