Objective To establish a rapid ,sensitive direct method for detecting Escherichia coli (E .coli) in the positive blood culture bottles by using the loop‐mediated isothermal amplification (LAMP) .Methods Based on the lacZ (accession number :M74750) gene sequence of E .coli in the gene sequence GenBank by the National Institutes of Health (NIH) ,4 specific primers (2 inner primers and outer primers) were designed ,the lacZ gene was amplified and the amplification reaction was optimized .The sen‐sitivity and specificity of this method were verified and its comparison with the traditional culture method was conducted .Results Sixty‐eight strains of E .coli were detected in 300 positive blood culture bottles by adopting the LAMP method .The designed prim‐ers had good sensitivity and good specificity for the amplification of E .coli .The sensitivity and specificity of LAMP all reached 100% compared with the traditional method ,but the traditional method needed 3 d to get the results ,while LAMP only needed 1 h . Conclusion LAMP is extremely rapid for detecting E .coli with low cost ,strong specificity and high sensitivity and is suitable for clinical application .

Objective To apply a one-step loop-mediated isothermal amplification(LAMP) assay for rapid detection of Escherichiaco-li .Methods According to the Genbank of Escherichiacoli lacZ(accession number:M74750) ,a set of four specific primers were designed for the Escherichiacoli LAMP assay and optimized the reaction .The results were judged with naked eyes and electrophoretic analysis .Results Genomic DNAs from 13 bacterial strains including Escherichia coli strains were amplified using LAMP ,and no amplicon was observed in the other bacterial strains .The detection limits of LAMP assay was 15 cfu/mL .Conclusion LAMP assay is extremely rapid ,cost-effective , specific and highly sensitive and has potential usefulness for clinical application .

Objective To investigate the effect of suppressor of cytokine signaling-1 (SOCS-1)on expression of monocyte chemoattractant protein-1 (MCP-1)in human glomerular mesangial cells (HMCs) under high concentration of glucose. Methods Stable transfections of HMC with pCR3.1 vector and pCR3. 1-SOCS-1 were performed with hpofectamine 2000, and cells were selected with geneticin. Cells were stimulated with low glucose (LG, 5.5 mmol/L), high glucose (HG, 30 mmol/L), LG plus mannitol (24.5 mmol/L) and AG490 (10 μmol/L). The protein expression levels of SOCS-1, signal transducer and activators of transcription 1,3 (STAT1, STAT3),p-STAT1 and p-STAT3 were observed by Western blotting. The protein synthesis of MCP-1, FN and type Ⅳ collagen in the supernatants of the HMCs were detected by ELISA and radioimmunoassay. The expression level of SOCS-1 and MCP-1 mRNA was measured by BT-PCR.Results HG induced the expression of SOCS-1 protein and mRNA in HMCs in time-dependant manner, peaked at 4 h, and gradually decreased to baseline at 24 h. Compared with low glucose control group, the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [(0.39±0.05) vs (0.16±0.02)]were significantly increased in HMCs under high glucose medium (P ＜0.01 ). Exposure of HMCs to high glucose conditions showed high concentration of MCP-1 [(459±67) ng/L vs (241±19) ng/L], fibronectin [(5.84±0.61) mg/L vs (3.41±0.31) mg/L]and type Ⅳ collagen [(16.45±2.30) μg/L vs (9.56±1.52) μg/L]in the supernatants (all P＜0.01).Overexpression of SOCS-1 inhibited the phosphorylation levels of STAT1 and STAT3 and the expression of MCP-1 mRNA [(0.34±0.04) vs (0.42±0.05)]in HMCs under high glucose condition (all P＜0.05). Compared with vector control group, the concentration of MCP-1 [(387±47) ng/L vs (463±56) ng/L], fibronectin [(4.61±0.57) mg/L vs (5.76±0.74) mg/L]and type Ⅳ collagen [(13.4±2.32) μg/L vs (17.1±2.57) μg/L]was decreased in supernatants of HMCs with SOCS-1 overexpression (all P＜0.05). Compared with HG group, the expression of MCP-1 mRNA (0.31±0.04) and the concentration of MCP-1 [(361±53) ng/L], FN [(5.46±0.71) mg/L]and type Ⅳ collagen [(15.2±1.97) μg/L]in supernatants were decreased in HMCs treated with AG490.Conclusion Overexpression of SOCS-1 inhibits overproduction of MCP-1 and ECM proteins in HMCs under high glucose conditions, which may be partly by regulating the phosphoryhtion of STAT1 and STAT3.

Aim To investigate the effect of sphingo-sine kinase 1 (SphK1 )on unilateral ureteral obstruc-tion(UUO)-induced tubulointerstitial fibrosis and ex-plore the possible mechanism.Methods The CD-1 mice were randomly divided into four groups:sham-op-eration group(Sham),PF-543 treatment control group (Sham +PF-543),model group(UUO)and PF-543 treatment group(UUO +PF-543).On 1 ,3,7 and 1 4 d after operation,eight mice were selected randomly from each group and sacrificed.The protein expressions of SphK1 ,mature TGF-β1 ,FN,ColⅠ,LC3,Beclin1 ,Atg5 and Atg1 2 were observed by Western blot.The histo-logical changes were examined by Masson′s trichrome stain.Immunhistochemistry was performed to measure the levels of expression of SphK1 ,FN and Col Ⅰ. Transmission electron microscope was used to observe the autophagic body.Results SphK1 expression and autophagy were both upregulated in a mouse model of kidney fibrosis induced by UUO. Meanwhile, in-creased mature TGF-β1 and deposition of extracellular matrix(ECM)were observed in tubulointerstitial areas compared with sham-operated mice.After intraperito-neal injection with the SphK1 specific inhibitor PF-543 in UUO mice,enhanced expression of SphK1 and acti-vated autophagy were significantly abrogated.Howev-er,aggravation of renal fibrosis was detected when SphK1 inhibitor PF-543 was applied to suppress SphK1 expression in UUO mice.Conclusion SphK1 activa-tion is renoprotective through the induction of autoph-agy in the pathogenesis of kidney fibrosis.

Objective To investigate mutation and deletion of genes mecR1 and mecI in clinical methicillin-resistant staphylococci isolates and study the mutation and deletion have effect on gene mecA expression and drug resistance phenotype.Metheods PCR was used to detecte gene mecA and the regulatory genes mecR1 and mecI in staphylococci which were separated from clinical specimen in 2006,then the sequence of gene mecI was determined and compared with the sequence obtains from pre-MRSA strain N315(GI:BA000018).Results Gene mecA was detected in 60 strains of Staphylococcus aureus,58 strains of Staphylococcus epidermidis and 37 strains of Staphylococcus heamolyticus,but gene mecA in 6 strains of Staphylococcus epidermidis and 4 strains of Staphylococcus heamolyticus were only amplified by primer mecA2-F/R and not by primer mecA1-F/R.The percentage of gene mecR1 exist in Staphylococcus aureus was higher than Staphylococcus epidermidis and Staphylococcus heamolyticus,but the percentage of gene mecR1 exist in Staphylococcus epidermidis was not higher than Staphylococcus heamolyticus.The mutation and deletion of gene mecI were often seen,the wild type mecI was only detected in 14 strains,the point mutation of nucleotice 202 was detected in 36 strains.Conclusions Gene mecA expression in Staphylococcus aureus could be chiefly induced by mecR1,but which in coagulase-ngeative staphylococci could be other factors.The mutation and deletion of mecI were universal phenomenon in clinical strains,there could be a mechanism for overcoming the repressing of resistance caused by mecI in staphylococci.

Aim To investigate the effects of fluvastatin on epithelial-myofibroblast transdifferentiation and activation of ERK1/2 in HKC cells stimulated by AGEs. Methods HKC are divided into four groups: control group, AGEs group, AGEs plus fluvastatin group and AGEs plus ERK1/2 MAP kinase inhibitor PD98059 group. Immunocytochemistry staining was used to detect expression of ?-SMA. The protein expressions of ?-SMA, E-cadherin, Col I, ERK1/2 and p-ERK1/2 were observed by Western blot. The protein synthesis of TGF-?1 in the supernatants of the HKC was detected by enzyme-linked immunoadsorbent assay (ELISA).?-SMA and E-cadherin mRNA were measured by reverse transcription and polymerase chain reaction (RT-PCR). Results Compared with those of control group,the expressions of ?-SMA protein and mRNA,and Col I were significantly increased in HKC cells with AGEs stimulation and there was high concentration of TGF-?1 in the supernatants. However, the expressions of E-cadherin protein and mRNA were decreased with AGEs stimulation. AGEs induced ERK1/2 phosphorylation in HKC in a time-dependent manner, being significant at 15 minutes and peak occured at 1 h. PD98059 and fluvastatin inhibited AGEs-induced activation of ERK1/2 and high expression of Col I and ?-SMA protein and mRNA, and reversed the expression of E-cadherin protein and mRNA induced by AGEs. Meanwhile, fluvastatin and PD98059 reduced the concentration of TGF-?1 in the supernatants of HKC with AGEs stimulation. Conclusions Fluvastatin inhibited AGEs-induced HKC epithelial-myofibroblast transdifferentiation and collagen I synthesis might be partly through blocking activation of ERK1/2.

Aim To investigate the effects of high glu-cose on cholesterol metabolism in renal tubular cells and the intervention of the anthocyanins. Methods HK-2 cells were grown in the DMEM medium supple-mented with 10% FBS and were divided into 5 groups:normal glucose group, high glucose group, mannitol group, C3G group and Cy group. Effect of anthocya-nins on cell viability was detected with MTT, and cho-lesterol accumulation was detected with Amplex Red Cholesterol Assay kit and Filipin staining. Expression of ABCA1 was detected with RT-qPCR and Western Blot. Results In compared with control groups, HG significantly promoted cholesterol mass inside the cell and decreased the cholesterol concentration in the me-dium after treatment for 24 h or 48 h. The levels of mRNA and protein of ABCA1 were detected with RT-qPCR and Western blot, and both were decreased in the presence of HG. Whereas treatment with C3G and Cy markedly attenuated HG-induced cholesterol mass inside the cell by up-regulating the expression of AB-CA1. Conclusions High glucose can reduce the ex-pressions of the ABCA1, and then decrease cholesterol efflux and increase the cholesterol accumulation in HK-2 cells. Anthocyanins can decrease cholesterol accu-mulation by up-regulating the expression of ABCA1.

AIM:To explore the change of antithrombin Ⅲ( AT-Ⅲ) in the patients with atherosclerotic cere-bral infarction .METHODS:Chromogenic substrate assay was used to measure the activity of AT-Ⅲ in 55 patients with atherosclerotic cerebral infarction and 55 healthy controls , and the correlation analysis was applied to determine the AT-Ⅲactivity with the severity of damage in central nervous system and general biochemical parameters .The levels of TNF-αand IL-6 in the plasma were detected by ELISA .Immunocomplex in the plasma was measured by enzyme immunoassay (EIA). The number and phenotype of the monocytes in peripheral blood were analyzed by flow cytometry .ELISA was also applied to determine the secretion of TNF-αand IL-6 from the monocytes after the stimulation of immunocomplex .The expression of AT-Ⅲin human brain vascular endothelial cells after the stimulation of TNF-αand IL-6 was observed by Western blotting . RESULTS:The activity of AT-Ⅲsignificantly decreased in the patients with atherosclerotic cerebral infarction , and nega-tively correlated with the damage degree of nervous system function , systolic pressure , diastolic pressure , glucose , choles-terol, triglyceride, low-density lipoprotein cholesterol and homocysteine , while positively correlated with high-density lipo-protein.In addition, the plasma levels of TNF-αand IL-6 increased significantly , accompanied with the enhancement of immunocomplex level .The numbers of CD14 + CD16 + and CD14 + CD32 + monocytes in peripheral blood were not changed , while CD14 +CD64 +monocytes increased obviously .The secretion of TNF-αand IL-6 by monocytes were signifi-cantly enhanced after stimulated with immunocomplex , while the protein expression of AT-Ⅲ in the human brain vascular endothelial cells was down-regulated after co-incubated with TNF-αor IL-6.CONCLUSION:Decreased AT-Ⅲactivity in the patients with atherosclerotic cerebral infarction is one of the risk factors of cerebral infarction , and related with the dis-ease severity .The production of pro-inflammatory cytokines through immunocomplex from CD 14 +CD64 +monocytes may be involved in the mechanism .Improvement of AT-Ⅲactivity may protect against cerebral ischemia .

Objective To investigate the effects of inhibiting miR-29 on growth,invasion and metastasis of pancreatic cancer PANC1 cells,and explore the potential mechanism.Methods Oligonucleotides inhibiting miR-29 (anti miR-29) and control oligonucleotides (miR NC) were used to transfect PANC1 cells to establish anti miR-29 PANC1 cells and miR NC PANC1 cells.Transient transfection of PUMA siRNA,E-cadherin siRNA or NC siRNA was used to construct cotransfected anti miR29 + PUMA-siRNA-PANC1 cells and anti-miR-29 + E-cadherin-siRNA-PANC1 cells.Number of colony formations was observed,cell survival was detected by MTT,cell apoptosis was measured by flow cytometry,cell invasion was detected by transwell chamber assay,and cell migration was detected by wound healing assay.Subcutaneous injection of anti miR-29 PANC1 cells was used to establish xenograft nude mice model,and venous injection of anti miR-29 PANC1 cells was used to establish lung metastasis nude mice model,and the subcutaneous and venous injection of PANC1 cells served as control.The growth of xenograft and the number of lung metastatic nodules were observed.TUNEL method was used to detect cell apoptosis in xenograft and immunohistochemical analysis was used to detect PUMA and E-cadherin in xenograft.Results The survival rate of PANC1,miR-NC-PANC1 and anti-miR-29-PANC1 cells was 100％,(96.8 ± 2.8) ％ and (24.4 ± 3.2) ％.The number of colony formation was (213 ± 36),(196 ± 28) and (37 ± 6) per 100 high power field.The number of transmembrane cells was (56.3 ± 9.6),(49.8-± 7.3) and (11.2 ± 3.4) per 400 high power field.The distance of cell migration was (260 ± 48),(247 ± 46) and (53 ± 7) μm.Cell apoptosis rate was (1.5 +0.9) ％,(2.6 + 0.9) ％ and (22.4 + 2.8) ％.There was statistically significant difference between anti miR 29 PANC1 cells and other PANC1 cells (P ＜0.05).The survival rate,apoptosis rate,transmembrane cells and migration distance of anti-miR-29 + PUMA-siRNA-PANC1 cells was (84.7 ± 10.9) ％,(1.3 ± 0.8) ％,(49.7 ± 6.4) per 400 high power field and (182 ± 36) μm,indicating that the effects of miR 29 inhibition on PANC1 cells were abolished (all P ＜0.05).The volume of the xenograft of PANC1 and anti-miR-29-PANC1 cells was (3 800 ±270) and (1 890 ± 160)mm3,the cell apoptosis rate was 0.93 ±0.14 and 8.26 ± 1.15,the number of metastatic lung lesions was (26.4 ± 6.5) and (8.6 ± 2.7),the PUMA positivity was (7.2 ±1.6) ％ and (43.8 ± 7.6) ％,E-cadherin positivity was (8.3 ± 3.6) ％ and (47.4 ± 5.7) ％,respectively.The xenograft volume and the number of metastatic lung nodules of anti miR29 PANC1 cells was obviously decreased or decreased,but cell apoptosis rate,PUMA positivity and E cadherin positivity were obviously increased,and the differences were all statistically significant (P ＜ 0.05).Conclusions Inhibiting miR-29 expression can decrease cell proliferation,migration and metastasis of PANC1 cells,and the potential mechanism may be associated with the upregulation of PUMA and E-cadherin.

Objective To determine the spectrum of conjunctival flora and the antibiotic susceptibility profiles of patients scheduled for penetrating intraocular surgeries.Methods A prospective case control study was performed.A total of 192 patients (192 eyes) scheduled for penetrating intraocular surgeries at the Fourth People's Hospital of Shenyang from February to August 2015 were enrolled.Samples from the conjunctival sac were collected before instillation of any ophthalmic solutions for both aerobic and anaerobic culture.The positive rate and bacterial spectrum were observed.Bacterial isolates were tested for antibiotic susceptibility to 7 commonly used ophthalmic antibiotics using automated drug resistance analyzing system.The research was approved by the Ethics Committee of the Fourth People's Hospital of Shenyang.Results Totally 91 strains were collected from 81 conjunctival samples during aerobic culture,the positive rate was 42.19％.Staphylococcus epidermidis was the most common microorganism (64.84％),followed by Staphylococcus lentus (7.69％) and Staphylococcus aureus (3.30％).Coagulatase negtive Staphylococcus (CNS) accounted for 80.22％ of the positively cultured aerobes.For anaerobic culture,a total of 28 strains were isolated from 28 conjunctival samples,the positive rate was 14.58％ Propionibacterium acnes was the predominant species (71.43％),followed by Finegoldia magna (10.71％).Majority of the CNS were sensitive to gentamycin and vancomycin,with resistance rates lower than 10％,but their resistance rate to erythromycin and ceftazidime was 87.67％ and 63.01％,respectively.Resistance rate of these CNS to levofloxacin,ciprofloxacin,and moxifloxacin was 42.47％,39.73％ and 17.81％,respectively.Multidrug resistance to at least 3 antibiotic classes was present in 38.36％ of the CNS.Conclusions Bacteria in the conjunctiva sac of preoperative patients are resistant to various ophthalmic antibiotics.To follow-up the bacterial distribution and antibiotic resistance is great meaningful in the prophylactic and treatment in ocular surgery-related infections.