Proteomic Study on the Effect of Wenshen Tongdu Formula （温肾通督方） on Spleen B Cells in Spinal Cord Injury Model Mice / 中医杂志

ObjectiveTo explore the effects and possible mechanism of Wenshen Tongdu Formula （温肾通督方， WTF） on spinal cord injury. MethodsThirty-six C57BL/6 female mice were randomly divided into sham operation group， model group and WTF group， with 12 mice in each group. The spinal cord injury model was established in the model group and the WTF group using the modified Allen's method， while in the sham operation group the spinal cord was only exposed. Since the 1st day after surgery， 50 g/（kg·d） of WTF solution was given to the WTF group by gavage， while 20 ml/（kg·d） of normal saline was given to the sham operation and model group by gavage， all for 14 days. Before surgery and on the 1st， 7th， and 14th days after surgery， the motor function of the mice was evaluated using the inclined plane test and hind limb motor function score （by BMS）. On the 3rd day after surgery， the nerve electrophy-siology was detected through electromyography and motor evoked potential； the spleen length was measured， and B cells in the spleen were sorted by magnetic beads； the differential expression of proteins were detected through proteomics technology； and the protein expression of mitochondrial outer membrane transport porin 20 （Tom20） and downstream cleaved caspase-3 in spleen B cells were measured using Western blotting. On the 14th day after surgery， MRI was used to observe the recovery of the spinal cord. ResultsCompared to those in the sham operation group at the same time， the BMS scores and subscores and the inclined plane test angle in the model group were reduced on the 1st， 7th and 14th days after surgery； the peak value of electromyogram and motor evoked potential were reduced， and the spleen length was shortened， while the expression of Tom20 and cleaved caspase-3 increased in splenic B cells increased （P＜0.05）. Compared to those in the model group at the same time， the BMS subscores on the 14th day and the angle of the inclined plane test on the 7th and 14th days after surgery increased in the WTF group； the peak value of electromyography and motor evoked potential， as well as the length of spleen increased， and the expression of Tom20 and cleaved caspase-3 decreased （P＜0.05）. The proteomics results showed that there were 100 differential proteins in the WTF group versus the model group， of which 37 were up-regulated and 63 were down-regulated. GO enrichment analysis showed that differential proteins mainly played their roles in oxygen binding， exogenous apoptosis negative feedback， zinc ion response， and oxygen transport. KEGG enrichment analysis showed that differential proteins were mainly concentrated in metabolic pathways， Huntington's disease， oxidative phosphorylation and other pathways. Subcellular localization showed that differential proteins were associated with mitochondria. Magnetic resonance imaging on the 14th day after surgery showed that the spinal cord structure of the mice in the sham operation group was intact， and the segments were clear， with normal spinal cord signal； the low signal area in the spinal cord injury area increased in the model group， and the spinal cord became significantly thinner； the injured segment had obvious depression in the WTF group， but the structure was more complete than that in the model group. ConclusionWTF may promote spinal cord injury repair by regulating immune function， and its mechanism may be related to inhibiting pyroptosis of spleen B cells.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Drug resistance mediated by outer membrane porin F (OmpF) and its regulatory gene of micF in Shigella strains / 中华微生物学和免疫学杂志

Objective To investigate the mechanism of drug resistance mediated by micF gene and outer membrane porin F ( OmpF) in Shigella strains. Methods Shigella strains were isolated from stool samples of patients who presented to the Second Hospital of Tianjin Medical University with acute diar-rhea in 2015. Antibiotic susceptibility test was performed to screen out the multidrug-resistant and non-multi-drug-resistant strains. The ompF gene was amplified by PCR. The micF and ompF genes at transcriptional levels in the two groups of strains were detected by quantitative real-time RT-PCR. Intracellular concentra-tions of ciprofloxacin in the two groups of Shigella strains were measured by automatic microplate reader. Re-sults According to the result of antibiotic susceptibility test, 13 strains that were resistant to 3 or more than 3 antibiotics were classified into the multidrug-resistant group, while the other 8 strains that were sensitive to all antibiotics used in this study or only resistant to 1 or 2 antibiotic were classified into the non-multidrug-re-sistant group. All of the 21 Shigella strains carried the ompF gene. Compared with the non-multidrug-resist-ant strains, the multidrug-resistant strains showed higher expression of micF gene, but lower expression of ompF gene. The differences in micF and ompF genes between the two groups were statistically significant. The result of correlation analysis suggested that there was a negative correlation between micF and ompF genes (r=-0. 244). The intracellular concentrations of ciprofloxacin in multidrug-resistant strains were low-er than those in the non-multidrug-resistant strains (P<0. 001). Conclusion The decreased expression of OmpF was one of the possible mechanisms of multidrug-resistance in Shigella strains. The micF gene was negatively related to the expression of OmpF. Moreover, the decreased intracellular concentrations of cipro-floxacin in multidrug-resistant strains might be related to the decreased expression of OmpF.

Effects of tetramethylpyrazine-2′-O-sodium ferulate on swelling of astrocytes and expression of AQP4 after oxygen-glucose deprivation/reoxygenation / 中国药理学通报

Aim To explore the effects of tetrameth-ylpyrazine-2′-O-sodium ferulate (TSF)on the swelling of astrocytes and the expression of AQP4 after oxygen gl-ucose deprivatio /reoxygenation(OGD /Reox).Methods Astrocytes were divided into 4 groups:control group, OGD /Reox group,Ozagrel group and TSF group.The effects of TSF on astrocytes were investigated 6,1 2,24 and 48 h after OGD /Reox.The cell injury was assessed by measuring LDH activity and MTT.The expression levels of AQP4 protein of astrocytes were detected u-sing Western blot.Results OGD /Reox induced obvi-ous cell swelling and significant reduction of LDH in astrocytes whereas TSF remarkably attenuated OGD-in-duced astrocyte swelling and LDH reduction (P group(P 0.05 ).Conclusion TSF can attenuate OGD-induced swelling of astrocytes through decreasing the AQP4 expression.

The dominant genotypes of porinⅠ gene of Neisseria gonorrhoeae isolates in Zhejiang Province and the construction of the prokaryotic expression system / 预防医学

Objective To analyze the dominant genotypes of porin Ⅰ gene encoding major outer membrane protein of Neisseria gonorrhoeae in Zhejiang Province and to construct the prokaryotic expression systems of porinⅠA and porinⅠB genes.Methods Based on the previous research,the porinⅠA and porinⅠB genes were sequenced after T -A cloning, and the dominant genotypes of porinⅠA and porinⅠB genes were analyzed.Then the prokaryotic expression systems of the dominant genotypes of porinⅠA and porinⅠB genes were constructed.Ten percent SDS -PAGE was applied to measure the output of PⅠA and PⅠB proteins after inducement by 0.5 mmol/L IPTG.Results All 5 porinⅠA isolates sequenced were serovar ⅠA -6.Of the 11 porinⅠB isolates sequenced,there were 5 serovarⅠB -3 isolates,3 serovarⅠB -3 /6 isolates,1 serovarⅠ B -6 isolate and 2 mutant isolates.Outputs of P Ⅰ A and P Ⅰ B expressed by the constructed prokaryotic expression systems PET -42 -PⅠA and PET -42 -PⅠB were as high as 30% and 20% respectively. Conclusion ⅠA -6 is the dominant genotypes of porinⅠA gene and ⅠB -3 is the dominant genotypes of porinⅠB gene in Zhejiang Province.Comparing to the porinⅠB gene,porinⅠA gene is more conserved.The prokaryotic expression systems with high efficiency of porinⅠA and porinⅠB genes were successfully constructed,which may be helpful in the further research of genetic engineering vaccine and clinical detection of Neisseria gonorrhoeae.

Serratia odorifera mediated enhancement in susceptibility of Aedes aegypti for chikungunya virus.

Background & objectives: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. Methods: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. Results: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. Interpretation & conclusions: tThe results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.

Análise molecular da expressão do fenótipo multi-droga resistente (MDR) em enterobactérias isoladas de amostras clínicas após exposição in vitro ao Imipenem / Molecular analysis of multi-drug resistant phenotype expression (MDR) in enterobacteria isolated from clinical specimens after exposure in vitro to imipenem

Após o surgimento e disseminação das ß-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos ß-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de ß-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a ß-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica

After emergence and broad dissemination of extended spectrum ß-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against ß-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other ß-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure

The carbapenem-resistance mechanism and homology analysis of four strains of Klebsiella pneumoniae isolated from different sites of one patient / 中国感染与化疗杂志

Objective To study the drug resistance mechanism and homologous relationship of four clinical strains of carbapen-em-resistant Klebsiella pneumoniae isolated from different sites of the same one patient.Methods Four carbapenem-resistant K .pneumoniae strains were isolated from blood,urine,sputum and pelvic drainage of a patient after cystectomy in clinical mi-crobiology lab of Xinhua Hospital in March 2012.① Modified Hodge test was used to detect the expression of carbapenemase.② PCR amplification assay and DNA sequencing were used to detect resistance-related genes.③ SDS-PAGE analysis was per-formed to analyze outer membrane porin components of the four carbapenem-resistant K .pneumoniae isolates.ERIC-PCR as-say was used to analyze the homology of these carbapenem-resistant isolates.Results Modified Hodge test demonstrated that all the four carbapenem-resistant K.pneumoniae expressed carbapenemase.KPC-2 gene was identified in all the four isolates by PCR test and DNA sequencing.SDS-PAGE analysis indicated an outer membrane porin alteration common to all the four iso-lates which is distinct from that of the strain sensitive to antibiotics.The identical DNA fingerprinting of the four strains was confirmed by ERIC-PCR analysis.Conclusions The antibiotic resistance mechanism of the four carbapenem resistant K.pneu-moniae associated with this case includes the expression of KPC-2 and altered outer membrane permeability induced by abnormal expression of outer membrane porin.These four strains belong to one common clonal group.

Carbapenem and cefoxitin resistance of Klebsiella pneumoniae strains associated with porin OmpK36 loss and DHA-1 β-lactamase production

Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.

Detection of beta-lactamase, porin-coding genes and linkage of KPC-ISKpn6 of the carbapenems-resistant Klebsiella pneumoniae / 中华微生物学和免疫学杂志

Objective To investigate the resistance-mechanism of the carbapenems-resistant Klebsiella pneumoniae isolated from clinical.Methods The clinical isolates of carbapenems-resistant Klebsiella pneumoniae from top three comprehensive hospitals of Nanjing area were examined by 40 beta-lactamase,porin-coding genes and linkage of KPC-ISKpn6 using PCR method,the PCR positive results were picked out for sequencing and sequencing BLAST search for comparison analysis.Results Twenty-four strains of carbapenems-resistant Klebsiella pneumoniae were detected,the positive rate of A beta-lactamase TEM-1 and SHV was 100％ (24/24),KPC-2 and LAP-2 was 95.8％ (23/24),45.8％ (11/24) respectively,and C beta-lactamase DHA was 4.2％ (1/24).Meanwhile,the positive detection rates of KPC-ISKpn6 linkage was 95.8％ (23/24),and the mutation rate of porin-coding genes ompK35 and ompK36 were up to 95.8％ (23/24) and 100％ (24/24).Conclusion High incidence of beta-lactamase TEM-1,SHV,KPC-2 and LAP-2 was found in the group of Klebsiella pneumoniae isolates,and carbapenems-resistant of which was primarily due to the high carrying rate of KPC-2 and the high mutation rate of porin-coding genes ompK35 and ompK36.The Insertion sequence ISKpn6 may be involved in the KPC-2 gene mediated-expression.

PBP1A, CarO and β-lactamase genes detection in a pandrug-resistant Acinetobacter baumannii / 中华临床感染病杂志

Objective To investigate resistant mechanisms of a pandrug-resistant Acinetobacter baumannii (js01) to β-1actams.Methods Strain js01 isolated from sputum sample of an inpatient from Ningbo First Municipal Hospital in December 2011 was confirmed by PCR amplifying and sequencing of gyrA and parC,and aligning with BLASTn.Thirty-three kinds of β-lactamase genes ( 13 kinds of class A,10kinds of class B,2 kinds of class C,8 kinds of class D),linkage detection of insertion sequences and β-lactamase genes,as well as outer membrane porin gene carO were analyzed by PCR.Genes encoding PBPI A were divided into three fragments,PCR amplified and bidirectional sequenced,and ligated to the full-length gene.Results Four kinds of β-lactamase genes were positive in js01:TEM-I,ADC-30, OXA-23 and OXA-66.Linkage detection of insertion sequences and β-lactamase genes showed that ISabal-ADC-30 and ISabal-OXA-23 were positive. When compared with sensitive strain (SDF) of Acinetobacter baumannii,sense mutations were found in carO gene of js01,and identity of amino acid sequence of carO gene between js01 and SDF was 76.0％ (189/249),and differences owed to loss of 3 amino acids.Sense mutations were also found in genes encoding PBP1A of js01,and identity of amino acid sequence of genes encoding PBP1A between js01 and SDF was 99.6％ ( 848/851 ),and differences owed to variations of 3amino acids.However,compared with three-dimensional structure of PBP1 A of SDF,PBP1 A of js01 lost 2helixes.Conclusion In strain js01,mutations of housekeeping genes ( genes encoding PBP1A and CarO),and genes producing β-lactamase mediated by mobile genetic elements,may play a key role in resistance to β-lactams.

Resistance of Klebsiella Pneumoniae clinical isolates: linkage of outer membrane proteins (omps) with production esbls

Three isolates of Klebsiella pneumoniae, collected from the University Hospital in Fortaleza, Brazil, were analyzed to determine their resistance to multiple antibiotics. The results of this study showed that the resistance of the clinically isolated bacteria is associated with the production of extended-spectrum beta-lactamases (ESLBs) and loss of outer membrane proteins.

Effect of irrigation with hypothermic artificial cerebral spinal fluid on aquaporin-4 expression in spinal cord following spinal ischemia-reperfusion in rabbits / 中华麻醉学杂志

Objective To investigate the effect of irrigation with hypothermic artificial cerebral-spinal fluid (aCSF)on expression of aquaporin-4(AQP-4) in the spinal cord following spinal ischemia-reperfusion (I/R) in rabbits.Methods Fifty-four adult male New Zealand white rabbits weighing 2.0-2.5 kg were randomly divided into 3 groups (n=18 each):group sham operation(S group); group I/R and group irrigation with hypothermic aCSF (FI group). Spinal I/R was induced by clamping the abdominal aorta below renal artery for 60 min. Hypothermic aCSF(25 X.)was infused at L4,5 interspace at a rate of 30 ml/h and drained from L7,8 interspace during spinal ischemia.Neurological function was evaluated at 4, 24, 48 and 72 h of reperfusion and scored (0=no hind limb activity, 4=hind limb function completely recovered) in 6 animals in each group. Six animals were sacrificed at 4, 24 and 72 h respectively in each group.The lumbar segment (L5-8) was removed for measurement of water content and AQP-4 protein expression (by immuno-histochemistry).Results Neurological function scores were significantly lower,water content was higher and AQP-4 expression smaller in group I/R than in group S. I/R-induced effects were significantly attenuated by irrigation of hypothermic aCSF. Conclusion Irrigation with hypothermic aCSF can ameliorate the spinal cord I/R injuries by up-regulation of AQP-4 expression.

Correlation of porin genes loss with resistance to carbapenems in Klebsiella pneumoniae / 中华临床感染病杂志

Objective To investigate the molecular mechanism of Klebsiella pneumoniae resistant to carbapenem. Methods The minimal inhibitory concentrations ( MICs) of the antimicrobial agents were determined by E-test. The 23 β-lactamase genes and 2 porin genes were amplified by polymerase chain reaction (PCR) , then the products were purified and their sequences were analyzed. Results The MICs of piperacillin, piperacillin/sulbactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefotaxime, cefepime and aztreonam to 5 strains of Klebsiella pneumoniae were all higher than 128 μg/mL, and those of imipenem or meropenem were higher than 32 μg/mL. All isolates carried blaTEM-1 and blaDHA-1 genes. Deletion of ompK35 and ompK36 were observed in Kp01 and Kp03, and the deletion of ompK35 was also observed in Kp02 and Kp05. Base insertion of ompK36 occurred in Kp02, Kp04 and Kp05. Compared with GenBank (GU945384) , ompK35 gene mutations of G→C at base 465 and T → C at base 466 in Kp04 lead to Gln to His substitution at position 155 and Tyr to its substitution at position 156, and it might be a new subtype. Conclusion The production of DHA-1 β-lactamase combined with the loss of OmpK36 or OmpK35 in porin genes may contribute to high-level carbapenem resistance in Klebsiella pneumoniae.

Porinas e suas ações imunomoduladoras dependentes de TLR2 / Porins and their immunomodulatory effects triggered by TLR2

Os micro-organismos podem infectar seu hospedeiro por diferentes vias, sendo a principal o trato respiratório. O reconhecimento pela mucosa dessas vias pode desencadear inibição da proliferação e bloqueio da entrada microbiana, assim como estimular resposta direcionada a memória imunológica para prevenir posteriores infecções. Alguns micro-organismo, como as bactérias Neisseria meningitidis e Neisseria lactamica, são capazes de modular a resposta imune de mucosa diretamente, ou por meio das células epiteliais respiratórias. Este trabalho propôs, então, a avaliação das porinas B provenientes destas bactérias como moduladoras da produção de IL-8 nas linhagens BEAS-2B e Detroit 562. Também foi avaliada a dependência deste estímulo ao receptor TLR2. Ambas as porinas se ligaram a TLR2 e por este receptor estimularam a produção de IL-8. O perfil de produção foi dependente da expressão de TLR2 pelas células. A porina lactâmica induziu menos IL-8 por regular negativamente a expressão de TLR2, mas sua afinidade pelo receptor se mostrou maior que a da porina meningocócica. As porinas são então moduladoras das células de mucosa, fato que somado a atividade adjuvante destas proteínas por via parenteral estimulou a avaliação destas como adjuvantes de mucosa. O modelo escolhido para a avaliação foi o de inoculação intranasal de camundongos, utilizando como antígeno o lipopolissacarídio pouco imunogênico de Franciscella tularensis atenuada (Ft-LPS). A análise foi baseada no título de anticorpos IgG e IgM séricos. A porina meningocócica se mostrou a mais imunogênica, mas por ser originária de patógeno acarreta maior risco biológico em sua produção. Para viabilizar a porina meningocócica como adjuvante, a mesma foi substituída por porina homóloga produzida de modo recombinante em Escherichia coli não patogênica. A porina recombinante foi avaliada pelo mesmo sistema in vivo e comparada a adjuvantes experimentais de ação conhecida (rCTB, QS-21 e ODN 1826). A porina apresentou...

Microorganisms can invade the host through many routes, specially the respiratory tract. The respiratory mucosa is responsible for recognition, inhibition, proliferation and entry blockade of microorganisms, besides incitation of immunological memory to prevent further infections. Some microorganisms, such as Neisseria meningitidis and Neisseria lactamica, can modulate the mucosa immune response directly or through stimulation of respiratory epithelial cells. The present work proposed the evaluation of porin B proteins, derived from these microorganisms, as modulators of IL-8 production on respiratory epithelial cell strains BEAS-2B and Detroit 562. TLR2 receptor dependency for the modulation was also evaluated. Both porins bounded to TLR2 and through this receptor were able to stimulate IL-8 production, whereas this profile was correlated with TLR2 expression. Lactamica porin (Nlac PorB) induced less IL-8 and TLR2 expression, also for a shorter period of time. The effect caused by Nlac PorB was attributed to TLR2 down regulated expression, since its binding affinity to the receptor is greater than meningococcal porin (Nmen PorB). Porins were therefore able to immune modulate mucosal cells, fact that allied with their parenteral adjuvant activity incited evaluation of porins as potential mucosal adjuvants. The model chosen for the evaluation was intranasal immunization of mice, using as the antigen a low immunogenic lipopolysaccharide extracted from attenuated Franciscella tularensis (Ft-LPS). The evaluation was based on IgG and IgM serum titers. After the immunization scheme, Nmen PorB induced higher IgG and IgM titers than Nlac PorB. Although Nmen PorB was more efficient, it comes from a pathogen. To overcome the risk of its production, it was replaced by recombinant porin (rPorB) produced by Escherichia coli. rPorB was evaluated by the same model and compared with well known experimental adjuvants (rCTB, QS-21 e ODN 1826). rPoB had the highest IgM and IgG...

Correlation between predominant pI genotypes,G120/A121 mutations and drug resistance / 中华流行病学杂志

Objective To analyze the predominant genotypes of outer membrane porin I (PI)and to determine the correlation between G120 as well as A121 mutations in PI proteins and drug resistance in Neisseria gonorrhoeae isolates in the local area.Methods A double PCR to simultaneously detect both pIA and pIB genes,was established in this study.The target amplification products were T-A cloned and then sequenced to determine the mutations at G120,A121 and the specificity of double PCR.By using acidity slip method and double agar dilution method,the β-lactamase production and resistance to six antibiotics of pIA~+ and pIB~+ gonoeoeeal isolates were detected.Results Double PCR could be used to accurately genotyping pI genes in all the tested gonococcal isolates with the sensitivity of 1 ng DNA template.In the 116 N.gonorrhoeae isolates,30.2%(35/116) were pIA~+ strains and 69.8%(81/136) were pIB~+ strains.All the pIA~+ strains presented G120D/A121G double mutations (88.6%) or A121G single mutation (11.4%).98.8% of the pIB +strains presented G120K/A121D (65.0%),G120K/A121G or G120N/A121D ( 13.8% ) double mutations,and G120D/N/K single mutation (21.3% ).34.5% (40/116) of the isolates produced β-lactamase,and the enzyme-produced rate (20%) in pIA~+ strains was significantly lower than that in pIB~+ strains (40.7%) with P＜0.05.No spectinomycin-resistant swains were identified but three ceftriaxoneresistant strains were presented.However,the resistance ratios to penicilin,terramycin,ciprofloxacin and azithromycin of all the isolates were as high as 75.0%-90.5%.100% and 71.4% of the pIA~+strains without β-lactamase production and with G120 and/or A121 mutations were sensitive topenicillin and terramycin,respectively.On the contrast,100% of the pIB~+ strains without β-1actamase production and with G120 and/or A121 mutations were resistant to both the two antibiotics.Conclusion The established double PCR method could be used for fast and accurate genotyping of N.gonorrhoeae pI genes.The N.gonorrhoeae strains prevalent in the local areas mainly possessed pIB gene.Both spectinomycin and ceftriaxone could still be chosen to treat gonorrhea.The resistance enhancement caused by G120 and/or A121 mutations to penicillin and tetramycin was only presented in pIB~+ gonococci.

The presence of high-level carbapenem resistance in Klebsiella pneumoniae is due to the combination of IMP-4 metallo-β-lactamase and porin OmpK36 deficiency / 中华检验医学杂志

Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.

Analysis of correlation between resistance of carbapenem agents in Pseudomonas aeruginosa and mutation and altered expression of oprD2 / 中华微生物学和免疫学杂志

Objective To investigate the impact on the resistance of carbapenem with the expression of OprD2 or OprD2 mutation in Pseudomonas aeruginosa. Methods One hundred and one clinical strains of Pseudomonas aeruginosa with MIC for imipenem ≥8 μg/ml were studied. MIC were determined by the broth microdilution method, and the antibiotics tested were imipenem(IPM ), biapenem( BPM), meropenem(MEM) and panipenem(PEM). The expression of the oprD2 gene in Pseudomonas aeruginosa were analyzed by real-time reverse transcriptase PCR(RT-PCR). For the Pseudomonas aeruginosa with normal expression of OprD2 and resistance to imipenem, full-length oprD2 gene was amplified by PCR and the products were sequenced. Results According to the result of the expression of oprD2 gene, 101 strains of Pseudomonas aeruginosa were divided into two groups: group1 with diminished expression of OprD2, and group2 with normal expression of OprD2. Comparing isolates with MIC of 4 kinds of carbepenem agents ≥ 16 μg/ml in two groups. Data showed the amount of OprD2 expression were different between two groups(P ＜0.01 or P ＜ 0.05). In group1, there are 28 isolates with MIC ≥ 16 μg/ml of all the 4 kinds of carbapenems, among which 25 isolates have obviously diminished expression of OprD2 ( ＜ 0.4). Negative correlations tendency appeared between the level of OprD2 transcription and MICs of 4 kinds of carbepenem agents in Pseudomonas aeruginosa. In group2, 16 strains with OprD2 mutation divided into 4 types according to the pattern of alteration. Compared with PAO1, these strains have increased MIC with different degree to IPM,BPM, MEM and PEM. Conclusion The deletion or diminished expression of OprD2 resulted in resistance to imipenem in Pseudomonas aeruginosa. The level of OprD2 transcription and antimicrobial activities for carbapenem agents proved to be highly correlated in Pseudomonas aeruginosa. The mutation of OprD2 in Pseudomonas aeruginosa probably decreased the sensitivity of carbapenem agents against Pseudomonas aeruginosa.

Stable Surface Expression of a Gene for Helicobacter pylori Toxic Porin Protein with pBAD Expression System / 华中科技大学学报（医学）（英德文版）

successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indi-rect immunofluorescence confirmed the expression of HopE on E. coli cell surface.

Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E.coli / 华中科技大学学报（医学）（英德文版）

Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.