Objective: To develop the ultra performance liquid chromatography tandem mass spectrometry ( UPLC-MS/MS ) for the determination of perfluorocarboxylic acids ( PFCAs ) in fish. Methods: The fish samples were extracted with tert-butyl methyl ether and purified by WAX columns. The WAX cartridges were rinsed with methanol and 25 mmol/L ammonium acetate, and the target compound residues were eluted with 0.5% ammonia methanol and then redissolved with 50% methanol aqueous solution after nitrogen blowing to nearly dry. Nine kinds of PFCAs were simultaneously quantified by UPLC-MS/MS with 1 mmol/L ammonium acetate-methanol solution as the mobile phase. Results: The extraction was separated well in UPLC BEH C18 column. There were good linear correlations of nine kinds of PFCAs in the range of 1.0-200.0 ng/mL, with the coefficients all more than 0.99. The limits of detection and quantification were 0.06-0.19 μg/kg and 0.19-0.62 μg/kg, respectively. The recovery rates were 70.08%-117.24% at different spiked levels ( 5.0, 25.0, 50.0 μg/kg ), and the relative standard deviations were 2.31%-19.68%. Conclusion Through optimizing the pretreatment conditions, the mobile phase of liquid chromatography and the detection conditions of mass spectrometry, the UPLC-MS/MS could meet the monitoring requirements of PFCAs in fish.

OBJECTIVETo study isolation and culture from SD rat bone marrow mesenchymal stem cells (rBMMSCs) and differentiate into endothelial-like cells (ELCs) with VEGF and bFGF.

METHODSIn vitro rBMMSCs were cultured with the method of percoll (1.073 g/ml) density centrifugation and adherence to plastic dishes. Then they were seeded in LG-DMEM supplemented with 10% fetal bovine serum. The relative biologic characteristics of rBMMSCs including cell morphology, phenotype, growth curve, cell cycle and ultrastructure were studied by inverted microscope, immunocytochemistry, flow cytometry, MTT method, transmission electron microscopy(TEM). The induced cells were identified by immunocytochemistry with CD31, CD34, CD144(VE-cadherin), FITC-UEA-1 and had ability in Dil-ac-LDL uptake and were observed under inverted microscope.

RESULTSThe morphology of rBMMSCs was spindle and has whirlpool. Growth curve of P4 showed that there was difference of latency, activity and flat periods. TEM showed that there were two of rMSCs , the small of both were infantile cells. GO/G1 phase of cell cycle was 95.67% and was suggested that most of cells were not in period of proliferation. The part differentiated cells demonstrated the characters of endothelial-like cells under inverted microscope and showed the expression of CD31, CD144, CD34, and possed the functions of endothelial-like cells staining double-positive for Dil-Ac-LDL and FTIC-UEA-1.

CONCLUSION1.073 g/ml percoll density centrifugation and cultured adherence is an effective approach to obtain rBMMSCs. In vitro, the cells have potential to differentiate into endothelial-like cells

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Vascular Endothelial Growth Factor A ; pharmacology

Brucellosis ; diagnosis ; drug therapy ; Child ; Child, Preschool ; Female ; Humans ; Male

Objective Laboratory tests of yeast, with their disadvantages of long identification, complicated operation, and low positive rate, cannot meet the needsof rapid diagnosis and timely treatment.This studyinvestigated the feasibility ofapplying matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI-TOF-MS) in rapid identification and clinical isolationof yeast strains. Methods A total of 120 clinical non-repetitive isolates were collected from clinical culture samples and identified by MALDI-TOF-MS and VITEK 2-Compact/API, respectively.The discordant results were resolved by ITS gene sequencing. Results Of the 120 yeast isolates analyzed, 117 (97.5%) were correctly identified at the species level by MALDI-TOF-MS, 1(0.8%) misidenti-fied, and 2 ( 1.7%) unidentified.ITS gene sequencing of the 3 isolates showed the coincidence rate to be 0( 0/3) for MALDI-TOF-MS and Vitek 2-Compact/API. Conclusion MALDI-TOF-MScan be used as a rapid, accurate, and inexpensive tool for the identification of clinical yeast strains.

Objective To explore the accuracy of Vitek 2 compact advanced expert system (AES) in indicating and analyze the carbapenemases-resisting Enterobacteriaceae phenotypes,and further investigate the methods to make up the AES.Methods 28 Enterobacteriaceae strains with Imipenem-Nonsusceptible by Vitek 2 compact,but AES suggested all production of carbapenemases were isolated.And imipenem susceptibility was determined by the disk diffusion method.Modified Hodge test (MHT) and the metallo-β-1actamase was detected by the double disk synergy method.Resistance genes were detected by the PCR amplification.Results ESBLs gene was amplified from all 28 selected strains,16 of which was detected KPC gene,and no strain of metallo-β-1actamases-producing bacteria.With carbapenemase gene detection as the gold standard,the accuracy of AES was 57.1％.Disc diffusion method detection accuracy rate of imipenem was 100％,and for 100％ of MHT accuracy.PCR amplification,MHT and the disk diffusion displayed the same result in detecting carbapenemases,but different with AES (x2 =10.08,P＜0.05).Conclusion The indications of the presence of carbapenemases using AES was not completely correct with a certain false-positive,and it is necessary to take other methods,such as disk diffusion or MHT methods,and improve the reliability of medicine-sensitivity tests.

Objective 30 Pseudomonasaeruginosa mechanism of resistance to quinolones.Methods For the determination of ciprofloxacin MIC by agar dilution method.Used PCR on DNA gyrase and topoisomerase Ⅳ,resistance genes gyrA,gyrB, parC and parE were amplified,and BLAST,to determine whether there was resistance to bits mutation point;using pulsed-field gel electrophoresis (PFGE)of these 30 strains homology analysis.Results The 28 bacterial strains gyrA gene ampli-fied fragment of 137 points were C→T mutation causes T83I;17 strains gyrB gene amplified fragment of 351 G→C lead to G466A;parC gene amplification 21 bacteria fragment 277 point increase with C→U mutation causes S87L change two differ-ent strains parE gene locus C→U mutation A425V and A473V cause change.PFGE results:30 Pseudomonas aeruginosa could be divided into six clones,Aclone 4,B clone 7,C clone 3,D clone 14,and two other single clones.Conclusion The tar-get mutant strains closely related to the epidemic clone type,the same changes in the same pop-type strains of drug targets, and proportional to the level of ciprofloxacin MICs value,the more the number of mutated genes,MICs value higher.GyrA gene most prone to mutation,the mutation was also the first to be discovered,more than any other target of the mutation mutations on binding of drugs and targets that would be the focus of concern.

OBJECTIVETo explore the intervention of baicalin on signal transduction and activating transcription factor expression of ulcerative colitis (UC) patients.

METHODSRecruited were UC patients at Outpatient Department of Digestive Disease, Inpatient Department of Digestive Disease, Center for Digestive Endoscopy of College City Branch, Guangdong Provincial Hospital of Traditional Chinese Medicine, and Southern Hospital affiliated to Southern Medical University from June 2010 to January 2011. They were assigned to the UC group (33 cases) and the diarrhea-predominant irritable bowel syndrome (IBS-D) group (30 cases). Another 30 healthy subjects were recruited as a healthy control group. Peripheral blood mononuclear cells (PBMCs) in vitro intervened by different concentrations baicalin were taken from UC patients. IL23R gene expressions in vitro intervened by different concentrations baicalin were detected using Q-PCR. Expressions of signal transducer and activator of transcription 4 (STAT4) , STAT6, phosphorylated-STAT4 (p-STAT4), and p-STAT6 were detected using Western blot. Serum levels of IFN-γ, IL-4, IL-6, and IL-10 were measured by ELISA. Effects of different concentrations baicalin on expressions of PBMCs, and levels of IFN-γ, IL-4, IL-10 of UC patients were also detected.

RESULTSCompared with the negative control group, 40 µmol baicalin obviously decreased IL23R gene expression of UC patients (P <0. 01). Compared with the healthy control group and the IBS-D group, p-STAT4/STAT4 ratios increased, p-STAT6/STAT6 ratios decreased, levels of IFN-γ, IL-4, IL-10 all increased in the US group (all P <0. 05). Compared with the negative control, 5 and 10 µmol baicalin groups, 20 and 40 moL baicalin obviously decreased p-STAT4/STAT4 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously increased p-STAT6/STAT6 ratios (all P <0. 05); 20 and 40 µmoL baicalin obviously lowered levels of IFN-γ and IL-4, and elevated IL-10 levels (all P <0. 05).

CONCLUSION40 µmoL baicalin could in vitro inhibit p-STAT4/STAT4 ratios, adjust p-STAT6/STAT6 ratios and related cytokines, thereby balancing the immunity and relieving inflammatory reactions of UC.

Activating Transcription Factors ; metabolism ; Anti-Inflammatory Agents, Non-Steroidal ; therapeutic use ; Blotting, Western ; Colitis, Ulcerative ; drug therapy ; metabolism ; Cytokines ; metabolism ; Flavonoids ; therapeutic use ; Humans ; Interleukin-10 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-6 ; metabolism ; Irritable Bowel Syndrome ; drug therapy ; metabolism ; Leukocytes, Mononuclear ; Medicine, Chinese Traditional ; Phosphorylation ; STAT6 Transcription Factor ; metabolism ; Signal Transduction

OBJECTIVEIt has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1).

METHODSIn vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected.

RESULTSAldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC.

CONCLUSIONStimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.

Aldosterone ; pharmacology ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor AP-1 ; metabolism

OBJECTIVETo evaluate the effects of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor (AT-1 receptor) blocker on the progression of rat pulmonary fibrosis induced by bleomycin A5.

METHODSTwenty-four male Wistar rats were randomized into pulmonary fibrosis model, perindopril treatment, losartan treatment and control groups. In the former 3 groups, pulmonary fibrosis was induced via intratracheal injection of bleomycin A5 (5 mg/kg), after which the rats in the perindopril and losartan groups received intragastric administration of the corresponding agents at the daily dose of 2 mg/kg and 10 m/kg, respectively. The rats in the control group had intratracheal injection of normal saline only. In the 4th week, the histological changes of the lung tissues were examined microscopically with Masson staining. Hydroxyproline content in the lungs was measured, and the protein expressions of AT-1 receptor, TGF-beta1 and IkappaBalpha were examined using Western blotting. DNA binding activity of NF-kappaB was analyzed with electrophoretic gel mobility shift assay (EMSA), and zymography was used to assess the activity of matrix metalloproteinase-2 and 9 (MMP-2, 9).

RESULTSBoth perindopril and losartan treatment significantly reduced the pulmonary fibrosis score, content of hydroxyproline, protein expression of TGF-beta1, DNA binding activity of NF-kappaB and MMP-2, 9 activity, and increased cytoplasmic protein expression of IkappaBalpha. Perindopril treatment lowered the protein level of AT-1 receptor.

CONCLUSIONPerindopril and losartan may inhibit bleomycin A5-induced pulmonary fibrosis in rats by reducing the protein expression of TGF-beta1 and suppressing the DNA binding activity of NF-kappaB and MMP-2, 9 activity.

Angiotensin II Type 1 Receptor Blockers ; therapeutic use ; Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Animals ; Bleomycin ; analogs & derivatives ; Blotting, Western ; Losartan ; therapeutic use ; Male ; NF-kappa B ; metabolism ; Perindopril ; therapeutic use ; Pulmonary Fibrosis ; chemically induced ; drug therapy ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Receptor, Angiotensin, Type 1 ; metabolism ; Transforming Growth Factor beta1 ; metabolism

AIM:To investigate the effect of toosendanin(TSN)on invasion and migration abilities of human ovarian cancer cells and the related mechanism.METHODS:The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations.The cell viabilty at 12,24,48,72 and 96 h after TSN treatment was measured by CCK-8 assay.Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells.The protein expression of nuclear factor-κB(NF-κB)p65,E-cadherin,N-cadherin,vimentin and Snail was determined by Western blot.RESULTS:TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells(P<0.05 ).Compared with control group ,the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly(P<0.05).In addition,the expression of NF-κB p65 and E-cadherin protein increased no-tably,followed with N-cadherin,vimentin and Snail protein decreased significantly(P<0.05).However,the inhibitor of NF-κB BAY11-7082 reversed the impact above.Compared with TSN group ,the migration and invasion abilities in TSN +BAY11-7082 group increased significantly(P<0.05).The protein expression of E-cadherin also decreased notably ,fol-lowed with the protein expression of N-cadherin,vimentin and Snail increased significantly(P<0.05).CONCLUSION:TSN inhibits the invasion and migration abilities of human ovarian cancer cells ,which is related to the inhibition of epitheli-al-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.