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Objective:To develop an ex vivo normothermic mechanical perfusion(NMP)and compare the effect of different portal perfusion pressures on attenuating hepatic injury from donor after cardiac death(DCD).Methods:All rat livers were subjected to in situ warm ischemia for 30 min after cardiac attest and thereafter stored for 8 h under cold preservation. Six livers were harvested and regarded as static cold storage(group CS, n=6). In experimental group, liver received an ex vivo dual NMP with oxygenated perfusion via hepatic artery for 2 h after cold storage. Hepatic injury was assessed and compared from perfused livers with full portal vein pressure(group M1, n=6)and low portal vein pressure(group M2, n=6). The evaluation parameters included perfusion flow, liver enzymes of perfusate, pathological changes by hematoxylin-eosin staining, Suzuki histological criteria, expression of activation markers of polymorphonuclear neutrophils and macrophages, myeloperoxidase (MPO)and CD68 by immunohistochemistry, level of malondialdehyde(MDA)and activity of superoxide dismutase(SOD). Results:In experimental group during NMP, perfusion flows tended to increase when portal pressures were stabilized in groups M1 and M2.Perfusion flow during NMP 60~120 min was significantly higher than during NMP 0~20 min.After NMP with full portal pressure, hepatic sinusoidal congestion, hepatocyte necrosis, steatosis and Suzuki criteria were lower in group M1 than those in group CS( P<0.05). Compared with group M1, lower hepatic injury was characterized with a lower change of liver enzymes in perfusate( P<0.05), a better histological evaluation( P<0.05), a lower level of MDA and a higher activity of SOD( P<0.05), lower expressions of CD68 and MPO ( P<0.05)and lower levels of TNF-α and IL-6( P<0.05)in perfused liver. Conclusions:The ex vivo dual NMP with oxygenated perfusion via hepatic artery mimics liver perfusion under the physiological conditions.NMP with a lower portal pressure can attenuate hepatic ischemia-reperfusion injury and confer a better protection against liver damage from DCD.
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Objective:To investigate the drug resistance and pathogenic mechanism of a uropathogenic Escherichia coli (UPEC) strain UPEC132 at the genome-wide level. Methods:The susceptibility of UPEC132 strain to 16 antimicrobial agents was determined by minimum inhibitory concentration (MIC) assay. The UPEC132 strain was genotyped by multilocus sequence typing (MLST). The three-generation sequencing platform was used to sequence and assemble the whole genome of the UPEC132 strain. Drug resistance and virulence gene function annotations were predicted by Prodigal software and screened by using genome database. Genome sequences of the UPEC132 strain and 23 other UPEC strains collected from GenBank were phylogenetically analyzed, and a phylogenetic tree was constructed by RAxML software.Results:The UPEC132 strain was resistant to ampicillin, ampicillin/sulbactam, tetracycline, erythromycin, chloramphenicol and cefazolin. Its genotype was ST10522 by MLST. The whole genome of the UPEC132 strain included one complete genome (chromosome) and two plasmid sequences. The sequence sizes of the chromosome and plasmids 1 and 2 were 5 234 468 bp, 117 139 bp and 101 356 bp, and the guanine-cytosine (GC) content was 50.48%, 49.05%, and 54.04%, respectively. There were 4 856, 140 and 116 genes annotated in the chromosome and plasmids 1 and 2, respectively. Drug resistance genes were mainly distributed in the chromosomal genome, mainly including the multidrug resistance efflux pump gene clusters. Only blaTEM-1 and tetG genes were carried in the plasmid 2. Virulence genes were also mainly distributed in the chromosome genome, including nine pilus adhesins, five iron uptake systems and three secretory toxins. Gene clusters encoding Afa and type Ⅳ fimbriae were located on plasmids 1 and 2, respectively. The phylogenetic tree showed that the UPEC132 strain was not in the same branch with either of the 23 UPEC strains. Conclusions:The UPEC132 strain belonged to ST10522, which was a newly named ST type of Escherichia coli and first reported at home and abroad. The genome-wide genetic information of the UPEC132 strain was fully revealed. The multidrug resistance genes and virulence genes carried by the UPEC132 strain were associated with its drug resistance and pathogenicity.
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Objective To analyze the characteristics of antibiotic resistance in group A Streptococ-cus ( GAS) strains isolated from children with scarlet fever in Tianjin in order to provide reference for clinical drug administration. -ethods GAS strains were collected from 2011 to 2016. A total of 276 isolates were analyzed by antibiotic susceptibility test and emm typing. Results All of the isolates were susceptible to penicillin, cefazolin and vancomycin, while 98. 2％ were susceptible to both chloramphenicol and levofloxa-cin. The resistance rates to azithromycin, erythromycin, clarithromycin, clindamycin and tetracycline were 97. 8％, 97. 1％, 94. 2％, 94. 2％ and 79. 3％. The concomitant resistance to erythromycin, azithromycin, clarithromycin, clindamycin and tetracycline was 73. 2％. The resistance rates of GAS strains isolated from different years to tetracycline, clindamycin, clarithromycin, erythromycin and azithromycin were significantly different. A statistically significant difference was found between the percentages of emm12 and emm1 strains resistant to tetracycline (84. 0％ vs 59. 5％, χ2=13. 820, P=0. 000). Conclusions The isolated GAS strains are sensitive toβ-lactams and highly resistant to macrolide antibiotics, clindamycin and tetracycline. Penicillin remains the preferred treatment for GAS infection and cephalosporins may be used as a substitute if the patient is allergic to penicillin.
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Objective To explore the role and mechanism of inducible nitric oxide synthase inhibitor 1 400W in alleviating ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells.Methods Human intrahepatic bile duct epithelial cells (HIBEC) in logarithmic phase were inoculated into culture plate at an appropriate density.The samples were randomly divided into control group (group C),ischemiareperfusion group (group I/R) and ischemia-reperfusion + 1 400W group (group I/R + 1 400W).Group C was cultured routinely;cells in I/R and I/R + 1 400W groups were placed in a three-gas incubator for 12h for simulating ischemia and then normal culture for 6h for simulating reperfusion.The I/R + 1 400W group had a final concentration of 100 μmol/L of 1 400W before ischemia and hypoxia.After reperfusion,cells and culture medium were collected,CCK 8 was used for detecting cell vitality,microplate method for detecting the content of lactate dehydrogenase (LDH) in culture medium,AnnexinV-FITC/PI double stain for detecting apoptosis level,Western blot for analyzing the expressions of endoplasmic reticulum stress (ERS)related protein cysteinyl aspartic acid protease 12 (caspase-12),glucose regulatory protein 78 (GRP78) C/EBP homologous protein (CHOP) and inducible nitric oxide synthase (iNOS).Results As compared with group C,cell viability significantly decreased in I/R and I/R+ 1 400W groups (53.8％ ± 2.3％ vs.100％,66.5 ％ ± 2.8 ％ vs.100 ％) (P＜0.05) while LDH increased markedly in cell culture medium (287.4 ±9.0U/L vs 120.2 ± 8.7U/L,212.0 ± 8.3U/L vs 120.2 ± 8.7U/L) (P＜0.05).Apoptosis accelerated markedly (41.5％±2.3％ vs5.2％±0.5％,32.7％± 1.8％ vs 5.2％±0.5％) (P＜0.05) and the expressions of caspase-12,GRP78,CHOP and iNOS spiked (P＜0.05);as compared with I/R group,cell viability of I/R+ 1 400W group rose while LDH,apoptosis level,caspase-12,GRP78 and CHOP declined in cell culture medium (P＜0.05).Conclusions 1 400W may alleviate ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells and its mechanism may be correlated with a suppression of endoplasrnic reticulum stress.
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Objective@#To investigate the etiological characteristics of Streptococcus pyogenes that caused scarlet fever from 2012 to 2016 in Tianjin.@*Methods@#The subjects were children diagnosed clinically as scarlet fever in Tianjin scarlet fever monitoring hospital from 2012 to 2016. The exclusion criteria were children with scarlet fever who were unable to cooperate with sampling. A total of 575 cases of children's swabs were collected. Biochemical methods were used to isolate and identify the bacteria of pharynx swab, and the PCR method was used to detect the emm genotyping and superantigen speA and speC, and the resistance of the strains to 10 antibiotics was measured by K-B paper method. We compared the carrying status of superantigen genes by different types of GAS and the resistance of all GAS to different antibiotics.@*Results@#There were 5 emm types (emm1/11/12/22/89). The dominant types were emm12 (52.9%, 100 strains) and emm1 (44.4%, 84 strains). The carrying rates of speA and speC genes were 21.7% (41 strains) and 76.7% (145 strains), respectively. The speA gene carrying rate of emm1 type GAS was 33.3% (28 strains), which were higher than that of emm12 (12% (12 strains)) (χ2=12.21, P<0.001). The speA and speC gene simultaneous carrying rate of emm1 type GAS was 27.4% (23 strains), which was higher than that of emm12 type (12% (12 strains)) (χ2=7.01, P=0.008). The percentages of the strains that were resistant to Azithromycin, Erythromycin, Clarithromycin, Clindamycin, Tetracyclin, Levofloxacin and Chloramphenicol were 96.8% (183 strains), 96.3% (182 strains), 92.1% (174 strains), 92.1% (174 strains), 73.0%(138 strains), 2.1% (4 strains) and 1.6% (3 strains), respectively. All isolates were susceptible to Penicillin, Cefazolin and Vancomycin, and there were statistical significance (χ2=953.28, P<0.001).@*Conclusion@#The dominant emm types causing scarlet fever are emm12 and emm1. The frequencies of speA and speC in emm1 and emm12 are different. S.pyrogenes in Tianjin were susceptible to penicillin, cefazolin and vancomycin, but highly resistant to the clindamycin, clarithromycin, erythromycin and azithromycin.
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Objective To study proteome variation between uropathogenic E. coil (UPEC)132, UPEC J96 and non-uropathogenic E. coli K-12 MG1655. Methods Two-dimensional gel electrophoresis(2-DE) was applied to compare the differential expression proteins between UPEC 132, UPEC J96 and non-uro-pathogenic E.coli K-12 MG1655. The differential expression proteins were digested in gel by enzyme. The mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting (PMF) were re-searched using the internet available database. Results The number of protein spots recognized from UPEC 132 was 466±11, significantly more than that of E. coli K-12 MG1655 (338±15) and UPEC J96 (382±12); there were 298 protein spots shared by the three E.coli strains, 56 protein spots shared by two UPEC strains, and 89 protein spots characterized by UPEC 132. Twenty-two differential expression or significantly increased expression protein spots, involved in virulence factors, metabolism and transportation, regulation of protein synthesis, biological oxidation and unknown functions, were successfully identified by MALDI-TOF-MS. Condusion The proteome from UPEC 132 and non-uropathogenic E. coli K-12 MG1655, or UPEC 132 and UPEC J96 was differentially expressed. It will provide important information on the pathogen-esis of UPEC 132.
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Objective To establish RT-PCR-RFLP method for studying the genotype of wild mea-sles virus strains isolated from Tianjin area from 2002 to 2008. Methods Isolations of measles virus were carried out by tissue culture method from urine and throat swab specimens collected from suspected cases. RNA were extracted from the virus specimens. The 594 bp fragment of C terminal of the N (nucleoprotein) gene was amplified by one-step RT-PCR, then the PCR products were digested with Bcn I , separated on agarose gel electrophoresis and then analyzed by the method of RFLP (restriction fragment length polymor-phism). In addition, above results were compared with DNA sequencing. Phylogenetic tree was plotted based on the results for the genetic relationship and distance analysis. Results Sixty-nine measles virus strains were isolated from 189 specimens from 2002 to 2008, of which the C terminals of N gene were all de-tected positive. Among the 69 strains of measles virus isolates, 98.55% (68/69) belonged to Hla sub-geno-type which was the predominant sub-genotype, and only one strain (1.45%) belonged to H1b sub-genotype by RFLP analysis which was in accordance with the results by DNA sequencing method. Phylogenetic tree analysis indicated the H1a sub-genotype measles virus strains should be further divided into 2 clades, and the variation fluctuated between 0.2% and 3.8%. There were transmission chains caused by different virus strains co-cireulation. Conclusion A genotype, H1a and H1b sub-genotype can be identified by RT-PCR-RFLP assay specically based on the restriction enzyme Bcn I .The RT-PCR-RFLP assay can be a rapid, simple, accurate and efficient method for large-scale surveillance of measles virus strains in China.
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Objective To establish a polymerase chain reaction method for the detection of the rcp virulence gene of L. pneumophila. Methods Sixteen strains of Legionella pneumophila, L. dumiffii, L. bozemanii and L. Longbeachae isolated from the cooling towers of the centralized air-conditioning systems in Tianjin form 2005 to 2007 were detected by polymerase chain reaction method using L. pneumophila rcp virulence gene-specific primer according to GenBank published nucleotide sequence. Results The 900 bp rcp genetic fragment was amplified in three strains of L. pneumophila, while others not. Conclusion The rcp virulence gene-based polymerase chain reaction method for the detection of L. pneumophila has been successfully established and is the foundation for the studies of Legionella virulence.
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Objective To construct the multi-epitope antigen(mea) gene of G2 glycoprotein of Hantavirus SEO type L99 strain.Methods The B cell epitopes was chosen and connected by three-peptide GPG after the amino acid sequence of G2 protein was analyzed and predicted by bioinformatical soft wares.The corresponding gene mea was constructed by overlap PCR,and cloned into the prokaryotic expression plasmid pET32a(+).Results The mea gene was successfully constructed by the five epitopes being chosen.The recombinant plasmid pET32a-mea was acquired by directional cloning.Conclusion The mea and its expression system E.coli BL21/pET32a-mea were constructed for the first time.The foundation was laid for the expression of the mea and its immunological applications.
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A method of streptomycin resistance screening was applied to improve t he productivity of Natamycin by Streptomyces gilvosporeus(ATCC13326) The sp ores treated with UV light were regenerated on agar plates containing 0 6?g/mL stre p tomycin 122 streptomycin resistant(str) mutants were obtained The Natamycin y iel ds of 13 mutants were higher than the original strain The mutants with high Na t amycin productivity were screened at a high frequency(10 6%) The highest one that demonstrated 1 46 times that of the original strain in Natamy cin productivity was obtained