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Objective:To evaluate the role of O-sialoglycoprotein endopeptidase (OSGEP) in hepatic ischemia-reperfusion injury (HIRI) and the relationship with oxidative stress in mice.Methods:Experiment Ⅰ Twenty-four SPF healthy male C57BL/6 mice, 12 wild-type and 12 OSGEP knockdown, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) by the random number table method: wild-type shamoperation group (Sham group), wild-type HIRI group (HIRI group), OSGEP knockdown+ sham operation group (Sham+ KD group) and OSGEP knockdown+ HIRI group (HIRI+ KD group). Ischemia-reperfusion model was prepared by blocking the hepatic artery and portal vein for 60 min followed by reperfusion in anesthetized animals, the blood vessels were only exposed without occlusion in Sham group and Sham+ KD group, and the blood vessels were clamped for 60 min followed by reperfusion in HIRI group and HIRI+ KD group. The mice were sacrificed after 6-h reperfusion to extract liver tissue samples for microscopic examination of histopathological changes (with an optical microscope after HE staining) which were evaluated using Suzuki score and for determination of the serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), level of reactive oxygen species (ROS) (using the DCFH-DA fluorescent probe method), contents of malondialdehyde (MDA) and glutathione(GSH) in liver tissues (using a colorimetric method) and expression of OSGEP (using Western blot). Experiment Ⅱ The well-growing AML12 cells were divided into 4 groups ( n=30 each) using a random number table method: control group (C group), oxygen-glucose deprivation/restoration (OGD/R) group, OGD/R+ OSGEP knockdown group (OGD/R+ KD group), and OGD/R+ OSGEP knockdown negative control group (OGD/R+ NC group). Group C was cultured under normal conditions. Group OGD/R was subjected to O 2-glucose deprivation for 6 h followed by restoration of O 2-glucose supply for 24 h in OGD/R group. In OGD/R+ KD group, stable transfection of AML12 cells with OSGEP knockdown was performed prior to the experiment, and the other procedures were the same as those previously described. The cell survival rate was measured by the CCK-8 assay, the release of lactate dehydrogenase (LDH) was measured, the DCFH-DA method was used to detect the levels of ROS, and the contents of MDA and GSH were determined using a colorimetric method. Results:Experiment Ⅰ Compared with Sham group, the expression of OSGEP was significantly down-regulated, the serum concentrations of AST and ALT, Suzuki score, levels of ROS and content of MDA were increased, and the GSH content was decreased in HIRI group ( P<0.05), and no significant change was found in each parameter in Sham+ KD group ( P>0.05). Compared with HIRI group, the serum concentrations of AST and ALT, Suzuki score, levels of ROS and content of MDA were significantly increased, and the GSH content was decreased in HIRI+ KD group ( P<0.05). Experiment Ⅱ Compared with group C, the expression of OSGEP was significantly down-regulated, the cell survival rate and GSH content were decreased, and the release of LDH, levels of ROS and content of MDA were increased in group OGD/R ( P<0.05). Compared with OGD/R group, the cell survival rate and GSH content were significantly decreased, and the release of LDH, levels of ROS and content of MDA were increased in OGD/R+ KD group ( P<0.05), and no significant change was found in each parameter in OGD/R+ NC group ( P>0.05). Conclusions:OSGEP plays an endogenous protective role in HIRI by inhibiting oxidative stress in mice.
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Objective:To evaluate the role of extracellular signal-regulated kinase (ERK)1/2 in glutamate-induced ferroptosis in PC12 cells.Methods:PC12 cells were divided into 6 groups ( n=21 each) using a random number table method: control group (C group), glutamategroup (Glu group), glutamate+ ERK1/2 over-expression group (Glu+ ERK1/2-OE group), glutamate+ ERK1/2 plasmid empty vector group (Glu+ Vec group), glutamate+ ERK1/2 knockdown group (Glu+ si-ERK1/2 group)and glutamate+ ERK1/2 SiRNA negative control group (Glu+ si-NC group). Cells were treated with glutamate at a final concentration of 6 mmol/L for 72 h in Glu group and with the equal volume of PBS buffer for 72 h in C group. Glu+ ERK1/2-OE group was transfected with ERK1/2 overexpression plasmid, Glu+ Vec group was transfected with plasmid empty vector, and Glu+ si-ERK1/2 group was transfected with ERK1/2 siRNA, Glu+ si-NC group was transfected with siRNA negative control for 48 h, and then glutamate at a final concentration of 6 mmol/L was added and cells were treated for 72 h. The cell viability, lactic dehydrogenase (LDH)activity and contents of glutathione (GSH), ferrous ions and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Mitochondrial membrane potential (MMP) and lipid reactive oxygen species (Lip-ROS) were measured by flow cytometry. Results:Compared with C group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, ferrous ions content, MDA content and Lip-ROS levels were increased in Glu group ( P<0.05). Compared with Glu+ Vec group, the cell viability, GSH content and MMP were significantly increased, and the activity of LDH, contents of ferrous ions and MDA, and Lip-ROS levels were decreased in Glu+ ERK1/2-OE group( P<0.05). Compared with Glu+ si-NC group, the cell viability, GSH content and MMP were significantly decreased, and the LDH activity, contents of ferrous ions and MDA, and Lip-ROS level were increased in Glu+ si-ERK1/2 group ( P<0.05). Conclusions:ERK1/2 is involved in glutamate-induced ferroptosis in PC12 cells.
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Objective@#To analyze the susceptibility and distribution of β-lactamase encoding and efflux pump genes as well as the inhibitory effect of efflux pump inhibitors on the carbapenem resistance of the carbapenem resistant Klebsiella pneumoniae without producing carbapenemase (CRKPPC). @*Methods@#One hundred and eight strains of carbapenem non-susceptible Klebsiella pneumoniae were collected from our hospital during 2012-2014. The strains producing carbapenemase were screened by Mastdiscs combi Carba plus disc system. For CRKPPC strains, the susceptibilities to antimicrobial agents were determined by micro-dilution broth methods. PCR and DNA sequencing technology were used to analyze the prevalence of β-lactamase encoding extended-spectrum β-lactamase (ESBL) and plasmid mediated AmpC genes as well as efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The inhibitory experiments were implemented by using carbonylcyanide-m-chlorophenylhydrazone (CCCP) and Reserpine to observe the role of efflux pumps on the carbapenem resistance. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to analyze the expression of outer membrane proteins OmpK35 and OmpK36. @*Results@#Twenty-six strains out of the 108 carbapenem non-susceptible Klebsiella pneumoniae were identified to be CRKPPC strains which displayed quite high resistance to β-lactam and high resistance to amikacin, gentamicin, levofloxacin and ciprofloxacin. Very good sensitivities were observed to tigecycline, polymyxin, ceftazidime/avibatan and aztreonam/avibatan. The high prevalence of bla CTX-M and bla SHV were also displayed with the prevalent rates being 76.9% and 57.7%, respectively. The loss of outer membrane proteins OmpK35 and OmpK36 with varying degrees was observed, among which 57.7% strains lost ompK35 and 19.2% strains lost OmpK36. More than 80% strains contained efflux genes including acrAB-tolC, kexD, kdeA, kpnEF and kpnGH. The results of inhibitory experiments showed that CCCP displayed quite obviously inhibitory effects on the carbepenem resistance whereas no inhibitory effect was observed for Reserpine. @*Conclusion@#Tigecycline and polymyxin could be used as the basis of combined drug for CRKPPC. The prevalence of AmpC, ESBLs and loss of OmpK35 and OmpK36 with varying degrees among these strains were observed, but the over-expression of efflux pumps should be the main mechanism of the carbapenem resistance in the Klebsiella pneumoniae strains, and efflux inhibitors may have potential value in treating the infections caused by these bacteria.
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Objective To investigate the antimicrobial resistance profile of the clinical isolates collected from selected hospitals across China. Methods Twenty-nine general hospitals and five children's hospitals were involved in this program. Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems. Results were interpreted according to CLSI 2017 breakpoints. Results A total of 190 610 clinical isolates were collected from January to December 2017, of which gram negative organisms accounted for 70.8％ (134 951/190 610) and gram positive cocci 29.2％ (55 649/190 610). The prevalence of methicillin-resistant strains was 35.3％ in S. aureus (MRSA) and 80.3％ in coagulase negative Staphylococcus (MRCNS) on average. MR strains showed much higher resistance rates to most of the other antimicrobial agents than MS strains. However, 91.6％ of MRSA strains were still susceptible to trimethoprim-sulfamethoxazole, while 86.2％ of MRCNS strains were susceptible to rifampin. No staphylococcal strains were found resistant to vancomycin. E. faecalis strains showed much lower resistance rates to most of the drugs tested (except chloramphenicol) than E. faecium. Vancomycin-resistant Enterococcus (VRE) was identified in both E. faecalis and E. faecium. The identified VRE strains were mainly vanA, vanB or vanM type based on phenotype or genotype. The proportion of PSSP or PRSP strains in the non-meningitis S.pneumoniae strains isolated from children decreased but the proportion of PISP strains increased when compared to the data of 2016. Enterobacteriaceae strains were still highly susceptible to carbapenems. Overall, less than 10％ of these strains (excluding Klebsiella spp.) were resistant to carbapenems. The prevalence of imipenem-resistant K. pneumoniae increased from 3.0％ in 2005 to 20.9％ in 2017, and meropenem-resistant K. pneumoniae increased from 2.9％ in 2005 to 24.0％ in 2017, more than 8-fold increase. About 66.7％ and 69.3％ of Acinetobacter (A. baumannii accounts for 91.5％) strains were resistant to imipenem and meropenem, respectively. Compared with the data of year 2016, P. aeruginosa strains showed decreasing resistance rate to carbapenems. Conclusions Bacterial resistance is still on the rise. It is necessary to strengthen hospital infection control and stewardship of antimicrobial agents. The communication between laboratorians and clinicians should be further improved in addition to surveillance of bacterial resistance.
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Objective To analyze the species classification and chracteristics of drug resistance and virulence in CTX-M producing Escherichia coli isolated from urine culture.Methods Escherichia coli cultured by urine were collected from our hospital during 2014,the ring disk diffusion test was implemented to determine the bacterial susceptibility,the EBLs determination test was used to analyze the bacterial EBLs producing situation;the enterobactoer duplicated gene spacer consensus sequency PCR(ERIC-PCR) was adopted to perform the genetic relation analysis;PCR was used to amplify the CTX-M encoding genes and multiple virulence genes iutA,ompT,fyuA,fdeC,fimH,traT,cvaC,pap,kpsMT,pAI,usp,aer,hlyA,cnf and chuA;the multiple PCR was used to analyze the species calssification of CTX-M-producing Escherichia coli;these strains of bacteria were classified as the CTX-M-producing group and non-CTX-M-producing group according to the results of CTX-M coding gene detection,the differences in the antibacterial drug resistance and virulence genes between the two gorups were performed the contrastive analysis.Results One hundred and sixty-two strains of E.coli by urine culture had no genetic correlation,among 126 EBLs positive strains,91 strains produced CT-M,in which 57 strains of CT-M producing Escherichia coli belonged to type D,and 116 strains belong to Type B2.The statistical analysis found that the drug resistance rate in the CTX-M-producing group was significantly higher than that in the non-CT-M producing group (except for imipenem),the prevalence of virulence genes including iutA,chuA and traT in the CT-M producing bacteria group was significantly higher than that in the non-CTX-M-producing group(P=0.001,0.006,0.000)Conclusion CTX-M-producing E.coli is main pathogenic bacterium of urinary infection in our hospital,its majority belong to type D with increased drug resistance,moreover has close correlation with virulence genes iutA,chuA and traA and is a pertential threat in clinical treatment of urinary infection.
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Objective To analyze the susceptibilities of Escherichia coli isolates collected from blood and the prevalence of ESBLs encoding genes.Methods A total of 121 Escherichia coli isolates collected from blood during 2012 were analyzed for antimicrobial susceptibilities by software of WHONET 5.6,the production of ESBLs was confirmed by confirmatory pheno-typic testing,PCR and DNA sequence were further implemented to analyze the ESBLs-encoding genes.Results 121 E.coli isolates displayed high resistance towards broad spectrum penicillin and 2nd or 3rd generation cephalosporins,levofloxacin and cotrimoxazole,with the resistance rates being more than 40%,susceptibilities to imipenem,piperacillin/tazobactam,ami-kacin were observed,with the resistance rates to be less than 12%,86(88.7%)out of 121 isolates were found to produce ESBLs.Among them,59.5% (72),38.8% (47)and 4.1% (5)were confirmed to carry blaCTX-M,blaTEM and blaSHV genes.Additionally,2(1.7%)isolates carried all the genes detected,30(24.8%)isolates carried both of blaCTX and bla-TEM,1(0.8%)isolate carried both of blaSHV andblaTEM.Conclusion Most of the E.coli isolates from the blood culture in Nanjing Gulou Hospital produce ESBLs,and displayed resistance towards most of the penicillins,cephalosporins and sin-gle amide antimicrobial agents should be chosen according to susceptibility results.
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Objective To analyze the drug resistance and homology of Acinetobacter baumannii (Ab) isolated from patients with explosive injury ,so as to explore the characteristics of drug resistance and prevalence of infection .Methods A total of 61 strains of AB isolated from clinical specimens of patients with explosive injury were collected .The antimicrobial susceptibility of these iso‐lates was detected by using K‐B test .All the strains were gene typed by using the pulsed field gel electrophoresis .Results The re‐sults of antimicrobial susceptibility test shown that the 61 isolates of Ab had high resistance rate ,and were multi‐drug resistant to common antibacterial agents ,except for tigecycline (the resistante rate was 11 .5% ) and minocycline (the resistante rate was 48 .0% ) .The 61 isolates of Ab were divided into 8 kinds of genotypes ,among which type A was the most prevalent one (25 strains) .Other genotypes were type B(10 strains) ,type C(6 strains) ,type D(4 strains) ,type E(8 strains) ,type F(3 strains) ,type G(4 strains) and type H(1 strain) .The isolates of Ab were with high homology .Conclusion Multi‐drug resistance is observed in strains of Ab isolates from patients with explosive injury .Clonal strains of AB may be disseminates among regions ,which indicates that high attention should be paid to these strains .
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Objective To explore the mechanisms of quinolones resistance in Acinetobacter baumannii and homology analysis a-mong the strains .Methods 25 strains of quinolones-resistant Acinetobacter baumannii isolated clinically were collected .Kirby-Bauer(K-B) detection was utilized to detect the sensitivity of conventional drugs ,and polymerase chain reaction (PCR) was em-ployed to detect quinolone resistance-related genes gyrA and parC which were verified by restriction enzyme digestion and sequen-cing ,repetitive extragenic palindrome(REP)-PCR was adopted to analyze the strain homology .Results Multiple resistances to 12 kinds of antibacterial agents were found among the 25 strains of Acinetobacter baumannii which were sensitive only to minocycline and amikacin ,with sensitive rates were 48 .0% and 32 .0% ,respectively ,and were all sensitive to polymyxin B [minimal inhibitory concentration(MIC)≤2 μg/mL] .gyrA and parC genes were found in the all strain .Mutation TCA→TTA(Ser→Leu) at coden 83 in gyrA gene existed in 25 strains ,mutation TCG→TTG(Ser→Leu) at coden 80 in parC gene existed in 23 strains ,mutation GAA→GGA(Glu→Gly) at coden 84 in parC gene existed in 2 strains .REP-PCR showed that the strains had high degree of homology . Conclusion Quinolone-resistant Acinetobacter baumannii has high degree of homology ,existing gyrA and parC gene mutations .
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Objective To assess the clinical value of pentraxin-3 (PTX-3) in diagnosis and survey of therapeutic effect for lung cancer.Methods The serum level of PTX-3,carcinoembryonic antigen (CEA),cytokeratin 19 fragment (CYFRA 21-1) were measured in 802 patients with lung cancer,462 with benign lung diseases and 522 healthy controls from multiple research centers,using ELISA and electrochemiluminescent assays.The clinical value of PTX-3 was assessed by comparing the area under receiver characteristic curves (AUC) with CEA and CYFRA21-1.The optimum cutoff value for diagnosis of lung cancer was investigated by maximizing the sum of sensitivity and specificity.By following-up,the serum level of PTX-3 was measured at 3 day,7 day,and 14 day in 61 lung cancer patients after surgical resection of lung cancer.Results In test group and validation,the serum levels of PTX-3 (g/L) are significantly higher in lung cancer group [9.21 (6.13-12.80),10.4(5.54-13.11)] than in benign lung diseases [5.28 (3.42-8.53),6.52 (3.84-7.89)] and in healthy controls [2.18 (0.54-5.44),2.44 (0.67-5.87)],[Z =8.161,14.118,(test group,all P ＜ 0.05) ;Z =9.832,17.595 (validation group,all P ＜0.05)].ROC curve showed the optimal cut-off values for PTX-3 was 8.03 g/L [AUC of 0.831,with a sensitivity of 76.1％ and specificity of 75.2％ in the test cohort; 0.828,71.3％,89.2％ in the validation cohort].Similar results were noted for early-stage lung cancer [0.764,79.1％,and 62.2％ in the test cohort; 0.744,71.3％,and 69.6％ in the validation].In the diagnosis of early-stage lung,the AUC and sensitivity and specificity of PTX-3 were 79.1％,0.764,71.3％ (test group),and 75.2％,89.2％,0.824 (validation group) significantly higher in these patients than CEA and CYFRA21-1.In small cell lung cancer,PTX-3 and NSE shared similar AUC differentiating LC from benign lung diseases and health controls.In following-up 61 lung cancer patients,PTX-3 levels before surgical resection of tumours [11.12(9.12-12.59)] was significant high than following 3 day after surgery(Z =4.32,P ＜0.01),and 14 day (5.12 ±2.54) vs.7 day (7.13 ±3.42) (t =2.143,P =0.023).The correlation between PTX-3 and CRP in LC,benign lung diseases,health control was 0.364,0.592,0.512 (all P ＜ 0.05).Conclusion Serum PTX-3 is a valuable biomarker of lung cancer and early-stage lung cancer with high sensitivity and specificity and improved identification of patients with lung cancer from those with non-malignant chronic lung diseases.
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A colony of mice with a unique trait of host resistance to tumorigenesis induced by transplantable cells has been established and studied by Cui Zheng et al.These spontaneous regression/complete resistance(SR/CR) mice possess a trait of resistance to a broad spectrum of tumors and can be transferred to cancer-sensitive mice,with an age-dependent spontaneous regression mediated mainly by innate leukocytes.This article reviews the discovery of the SR/CR mouse model and related study of its anti-cancer mechanism.