Parasitosis y antígenos TF, Tn y sTn / Parasitosis and TF, Tn and sTn antigens

Resumen Las investigaciones realizadas establecen una relación entre los epítopos T, Tn y sTn y las enfermedades parasitarias. Estos epítopos se expresan en un alto porcentaje de tumores epiteliales e inicialmente fueron relacionados con el síndrome T, caracterizado por trombocitopenia, leucopenia y anemia hemolítica. Se ha identificado la expresión de Tn en varios carcinomas, aunque los eventos asociados a su exposición en éstos parecen ser diferentes de los observados en el síndrome Tn. Diversos estudios comunicaron que estructuras asociadas a tumores, tales como los antígenos Tn y sialil-Tn, se expresan en algunos protozoarios y helmintos, y plantearon numerosos interrogantes a nivel de la interacción parásito-hospedador, de la glicobiología parasitaria y de las eventuales relaciones entre la biología de algunos parásitos y las células cancerígenas. Los hematíes son poliaglutinables cuando son aglutinados por casi todas las muestras de suero humano normal. Algunas formas de poliaglutinidad se deben a la exposición del determinante antigénico TF, mediante la eliminación del ácido N-acetilneuramínico, por la acción de neuraminidasas bacterianas o virales, aunque en los últimos años se ha comunicado el desenmascaramiento de este antígeno críptico eritrocitario por Ascaris lumbricoides y Trichinella spiralis. Debido a la importancia clínica de la activación T, se destaca la necesidad de estudiar la exposición del antígeno críptico TF en todos los parásitos cuyo hábitat sea la sangre, o bien en aquellos cuyos ciclos de vida comprendan una migración por el torrente circulatorio, pues su desenmascaramiento puede ocasionar autoaglutinación y/o hemólisis.

Abstract The investigations carried out establish a relationship between the T, Tn and sTn epitopes and the parasitic diseases. These epitopes are expressed in a high percentage of epithelial tumors, and they were initially related to the T syndrome, characterised by thrombocytopenia, leukopenia, and hemolytic anemia. The expression of Tn has been identified in several carcinomas, although the events associated with its exposure appears to be different from those observed in the Tn syndrome. Various studies report that tumor-associated structures such as Tn and sialyl-Tn antigens are expressed in some protozoa and helminths, raising numerous questions at the level of parasite-host interaction, parasitic glycobiology and eventual relationships between the biology of some parasites and cancer cells. Red cells are polyaglutinate when agglutinated by almost all normal human serum samples. Some forms of polyaglutinity are due to the exposure of the antigenic determinant TF, through the elimination of N-acetylneuraminic acid, by the action of bacterial or viral neuraminidases, although the unmasking of this erythrocyte cryptic antigen by Ascaris lumbricoides and Trichinella spiralis has been reported in recent years. Due to the clinical importance of T activation, the need to study the exposure of the cryptic TF antigen in all parasites whose habitats are blood or whose life cycles include migration through the circulatory stream is highlighted, since its unmasking can cause autoagglutination and/or hemolysis.

Resumo As investigações realizadas estabelecem uma relação entre os epítopos T, Tn e sTn e doenças parasitárias. Esses epítopos são expressos em alta porcentagem de tumores epiteliais e foram inicialmente relacionados à síndrome T, caracterizada por trombocitopenia, leucopenia e anemia hemolítica. A expressão de Tn foi identificada em vários carcinomas, embora os eventos associados à sua exposição neles pareçam ser diferentes dos observados na síndrome Tn. Vários estudos relatam que estruturas associadas a tumores, tais como antígenos Tn e sialil-Tn, são expressos em alguns protozoários e helmintos, levantando inúmeras questões no nível da interação parasita-hospedeiro, glicobiologia parasitária e eventuais relações entre a biologia de alguns parasitas e células cancerígenas. Os glóbulos vermelhos são poliaglutináveis quando são aglutinados por quase todas as amostras de soro humano normal. Algumas formas de poliaglutinidade são devidas à exposição do determinante antigênico TF, pela eliminação do ácido N-acetilneuramínico, pela ação de neuraminidases bacterianas ou virais, embora nos últimos anos tenha sido relatado o desmascaramento desse antígeno eritrocitário críptico por Ascaris lumbricoides e Trichinella spiralis. Devido à importância clínica da ativação T, destaca-se a necessidade de estudar a exposição do antígeno críptico TF em todos os parasitas cujo habitat seja o sangue ou naqueles cujos ciclos de vida incluam migração pela corrente circulatória, uma vez que seu desmascaramento pode causar autoaglutinação e/ou hemólise.

Screening of new virulence genes of high virulence Klebsiella pneumoniae by Tn-seq / 中华微生物学和免疫学杂志

Objective:To construct a transposon mutation library and screen new virulence genes of hypervirulent Klebsiella pneumoniae(hvKp). Methods:The transposon mutation library was constructed and treated with human serum. The changes in the abundance of the genes of library mutant strains were analyzed by Transposon sequencing (Tn-seq). Besides, KEGG (kyoto encyclopedia of genes and genomes) annotation and enrichment analysis were performed on the screened genes.Results:A total of 405 genes were screened out according to the abundance of the genes in the library treated with human serum was 20% lower than that without treated, and 351 genes, 86.7% of these genes were conserved in HS11286, NJST258_1, NTUH-K2044 and RJF293. Ten genes existed in strains NTUH-K2044 and RJF293 with high virulence, while these genes were absent in HS11286 and NJST258_1 with low virulence. The mutants with genes such as glycosyl transferase gene wzy, aggregator protein gene wzi and capsule transporter gene wza, which belong to the capsule polysaccharide gene clusters, could not be detected after serum treatment. The abundance of iron carriers gene clusters such as aerobacterin and salmonellin in each library changed less than one time. KEGG annotation results showed that most annotated genes were involved in amino acid metabolism, cofactor and vitamin metabolism, carbohydrate metabolism, etc. Conclusions:Tn-seq is a reliable method to screen functional genes. In this study, 405 candidate virulence genes of hvKp were successfully screened out, providing an experimental basis for further research on the function and regulation mechanism of new virulence genes of hvKp.

Effect of Triptonide on Proliferation and Apoptosis of SHI-1 Cells / 中国实验方剂学杂志

Objective:This study aims to investigate the effect of triptonide （TN） on proliferation， cell cycle， apoptosis and expressions of apoptosis-related proteins of human acute monocytic leukemia（AML） cell line SHI-1， and to explore its possible mechanism of action. Method:The thiazolyl blue （MTT） colorimetric assay was applied to detect the inhibitory effect of 20，40，80，160，320 nmol·L^-1 triptonide on the proliferation of SHI-1 cells for 48， 72 h. Changes in SHI-1 cell cycle before and after triptonide treatment were detected by flow cytometry propidium iodide （PI） simple staining， and changes in SHI-1 cell apoptosis before and after triptonide treatment were detected by flow cytometry with AnnexinV/PI double staining. Western blot was applied to detect the protein expression of cysteine protease （Caspase）-3， Caspase-8 and nuclear transcription factor kappaB（NF-<italic>κ</italic>B） in SHI-1 cells before and after treatment with 80， 160 nmol·L^-1triptonide. Result:Compared with the blank group， 40，80，160，320 nmol·L^-1 triptonide significantly inhibited the proliferation of SHI-1 cells（<italic>P</italic><0.01） in a dose-dependent manner for 48， 72 h， while 160， 320 nmol·L^-1 triptonide induced apoptosis of SHI-1 cells（<italic>P</italic><0.01） for 48， 72 h， and 160 nmol·L^-1 triptonide could decrease the S phase ratio of SHI-1 cells（<italic>P</italic><0.01）. In addition， compared with the blank group， 80，160 nmol·L^-1 triptonide induced the downregulation of NF-<italic>κ</italic>B significantly（<italic>P</italic><0.01）， 160 nmol·L^-1 triptonide induced the downregulation of Caspase-3， Caspase-8 significantly（<italic>P</italic><0.01）. Conclusion:Triptonide can inhibit the proliferation and induce apoptosis <italic>in vitro</italic> of SHI-1 cells， which may be related to the reduction of the cells in S phase proportion by triptonide， and the downregulation of the expression levels of Caspase-3， Caspase-8 and NF-<italic>κ</italic>B proteins.

Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin / 中国中药杂志

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing / 南方医科大学学报

OBJECTIVE@#To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data.@*METHODS@#Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis.@*RESULTS@#Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.@*CONCLUSIONS@#Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.

Diversity of metallo-b-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region

BACKGROUND The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES The present study aimed to verify the occurrence of metallo-β-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

Estudo genotípico e fenotípico de bacilos Gram-negativos produtores de carbapenemase do tipo New Delhi metalo-ß-lactamase / Genotypic and fenotypic study of Gram-negative bacilli producers carbapenemase type New Delhi metallo--ß-lactamase

Os carbapenêmicos são os antimicrobianos mais amplamente utilizados no tratamento empírico de infecções graves por bacilos Gram-negativos. A pressão seletiva gerada pelo uso desses antimicrobianos ao longo das últimas três décadas contribuiu para a disseminação de enterobactérias e Gram-negativos não fermentadores produtores de carbapenemases, particularmente as do tipo KPC e NDM. Os genes que codificam essas enzimas usualmente estão localizados em plasmídeos e/ou transpósons. A hipótese atualmente mais aceita é que o gene blaNDM-1 seja uma quimera criada em Acinetobacter baumannii. A NDM-1 foi descrita em paciente proveniente da Índia e subsequentemente evidenciou-se sua ampla disseminação nesse país. A epidemiologia que tem sido observada nos casos detectados na Europa e Estados Unidos tem sido viagem à Índia, ou seja, sem casos autóctones. No Brasil, os primeiros casos foram identificados no Rio Grande do Sul, e a seguir no Rio de Janeiro e em São Paulo. Diferentemente dos casos da Europa e América do Norte, os casos do Brasil não tem relação epidemiológica com a Índia. O sequenciamento integral dos plasmídeos e cromossomos albergando o gene blaNDM permitirá entender como ocorre a disseminação desse mecanismo de resistência no Brasil. Para isso, foi avaliado o perfil de susceptibilidade dos isolados, bem como a capacidade conjugativa e clonalidade. Das vinte e oito amostras utilizadas neste trabalho, treze delas pertencem à espécie Enterobacter hormaechei, uma à espécie Citrobacter freundii, sete à espécie Escherichia coli, quatro à Klebsiella pneumoniae e três ao gênero Acinetobacter spp. Os primeiros isolados incluídos neste estudo (Escherichia coli e Enterobacter hormaechei produzindo NDM-1) foram isolados em agosto de 2013, de uma mesma amostra de swab retal de um paciente do Rio de Janeiro que nunca viajou para o exterior. O sequenciamento completo do DNA plasmidial utilizando a plataforma Illumina e a anotação de ambos os plasmídeos albergando o gene blaNDM-1 revelou que estes pertencem a grupos de incompatibilidade diferentes, IncFIIK (E. hormaechei) e IncX3 (E. coli), e abrigam um novo transpóson composto designado Tn3000. A comparação da sequência nucleotídica do Tn3000 com aquelas disponíveis no GenBank evidencia que a mesma estrutura está presente em plasmídeos de isolados da cidade de Porto Alegre e também em diferentes continentes. As espécies de Acinetobacter (A. radioresistens, A. ursingii e A. guillouiae) isoladas em São Paulo e Porto Alegre, possuem o gene blaNDM-1 albergados em um mesmo plasmídeo não tipável de 41.087 pb. A avaliação da clonalidade dos isolados de Enterobacter hormaechei "subsp. oharae" mostrou dois perfis diferentes através da técnica de PFGE, sendo que todos os microrganismos foram isolados de um surto no mesmo hospital no Rio de Janeiro. Isolados de Klebsiella pneumoniae de uma mesma paciente internada em hospital em Salvador, de sítios distintos - swab retal, hemocultura e urina, em ordem cronológica - obtiveram o mesmo perfil clonal pela técnica de PFGE. O mesmo ocorreu com três isolados de Escherichia coli, de um mesmo paciente do Rio de Janeiro, em amostras de swab retal. Os achados deste estudo evidenciam que no Brasil, Nepal, Marrocos e Índia há uma disseminação do gene blaNDM-1 mediada por um novo elemento móvel designado Tn3000 em enterobactérias. A detecção de um mesmo plasmídeo em diferentes espécies de Acinetobacter evidencia que neste gênero bacteriano, no Brasil, a disseminação do gene blaNDM-1 ocorre por conjugação.

Carbapenems are the antimicrobials most widely used in the empirical treatment of severe infections caused by Gram-negative bacilli. The selective pressure generated by the use of these antibiotics over the last three decades has contributed to the spread of enterobacteria and Gram-negative non-fermenting producing carbapenemases, mainly KPC and NDM. Genes encoding these enzymes are usually located in plasmids and/or transposons. Currently the most accepted hypothesis is that the blaNDM-1 gene is a chimera created in Acinetobacter baumannii. The NDM-1 was described in a patient from India and subsequently was reported to be broadly disseminate in this country. The epidemiology that has been observed in cases detected in Europe and United States is traveling to India, but no autochthonous cases. In Brazil, the first cases were identified in Rio Grande do Sul, and then in Rio de Janeiro and São Paulo. Differently from the cases described in Europe and North America, the cases from Brazil have no epidemiological link with India. The complete sequencing of plasmids and chromosomes harboring blaNDM gene will understanding how the dissemination of this resistance mechanism in Brazil occurs. In this work we will be evaluate the susceptibility profile of the isolates, and their conjugal capacity and clonality. Of the twenty-eight samples used in this study, thirteen of them belong to the species Enterobacter hormaechei, one to Citrobacter freundii, seven to Escherichia coli, four to Klebsiella pneumoniae and three to the genus Acinetobacter sp. The first two isolates included in this study (Escherichia coli and Enterobacter hormaechei) were isolated in August 2013, from the same rectal swab sample from a patient from Rio de Janeiro that never traveled abroad. Complete sequencing of plasmid DNA using Illumina platform and annotation of both plasmids harboring the blaNDM-1 gene revealed that they belong to different incompatibility groups, IncFIIK (E. hormaechei) and IncX3 (E. coli), and are harbor to a new transposon designated Tn3000. The comparison of the Tn3000 nucleotide sequence with those available at GenBank shows that the same structure is present in plasmids from other Porto Alegre and also in different continents. The Acinetobacter species (A. radioresistens, A. ursingii and A. guillouiae) isolated in São Paulo and Porto Alegre, have the blaNDM-1 gene harbored in a single non-typing plasmid of 41,087 bp. The evaluation of clonal relationship of Enterobacter hormaechei "subsp. oharae" showed two different profiles by PFGE technique; of note all microorganisms were isolated from an outbreak in the same hospital in Rio de Janeiro. Isolates of Klebsiella pneumoniae from a single patient hospitalized in Salvador, from different anatomical sites - rectal swab, blood culture and urine, in chronological order - obtained the same clonal profile by the PFGE technique. The same occurred with three Escherichia coli isolates, from the same patient from Rio de Janeiro, in swab rectal strains. Our findings suggest that in Brazil, Nepal, Morocco and India there is a spread of blaNDM-1 gene mediated by Tn3000 in enterobacteria. The detection of a same plasmid in different species of Acinetobacter shows that in this bacterial genus, in Brazil, the dissemination of the blaNDM-1 gene occurs by conjugation.

Improvement of pyruvate production by Escherichia coli via pathway engineering and Tn5 transposon mediated mutagenesis / 生物工程学报

To develop a high-yield pyruvate strain, we first engineered a pyruvate-producing Escherichia coli KLPP from wild-type E. coli MG1655 by blocking the pathways for byproduct formation via gene knockout. Then, we built a library of mutant containing 7 197 monoclones by using the pUT Mini-Tn5 transposon vector for random mutagenesis with E. coli KLPP. We developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader. After two-round screening we successfully obtained six mutants with increased pyruvate titer using this method, the titer of pyruvate was increased by 38%, 31%, 19%, 28%, 44% and 14%, respectively. The position of transposon insertion was determined by whole genome re-sequencing, and the gene locus possibly influencing pyruvate production was analyzed, which laid the foundation for subsequent strain improvement by metabolic engineering.

Evaluation of the analytical performance of AccuTnI+3 assay and establishment of the regional 99th percentile from an apparently healthy Chinese population / 中华检验医学杂志

Objective To validate the analytical performance of a cardiac troponin I(cTnI)assay AccuTnI+3 on chemiluminescnet analyzer DXI800 and Access2;and to establish the 99th percentile of cTnI in an apparently healthy Chinese population.Methods The subjects are composed of 1 369 apparently healthy people and 20 acute myocardial infarction(AMI)patients from Wuhan Asian Heart Hospital and Fuwai Hospital from October 2014 to June 2015.The healthy people include 680 males and 689 females;with 340 subjects aged 18-30,674 subjects aged 31-64, and 355 subjects aged ≥65.The detection limits and imprecision of AccuTnI +3 assays were validated according to CLSI EP 15-A2 and EP17-A2 documents;the same samples were analyzed on DXI800 and Access2 to assess the consistency between the two analyzers using Bland Altman plot and Passing-Bablok regression.The correlation between different sample types (lithium heparin plasma, EDTA plasma & serum)were assessed using linear regression analysis.The lithium heparin plasmasamples from 1 369 apparently healthy people were analyzed to calculate the 99th percentile of cTnI.The cTnI concentrations were compared among age and sex groups.The 99th percentile of cTnI were also calculated for each group.The detection rate of cTnI in apparently healthy people was calculated using SPSS23.0.Results The limit of blank(LoB), limit of detection(LoD), and limit of quantification(LoQ)where CV%=10% were 0.007 ng/ml,0.010 ng/ml and 0.016 ng/ml on DXI800;0.008 ng/ml,0.012 ng/ml and 0.026 ng/ml on Access2,respectively.The cTnI measurements on DXI800 and Access2 were consistent and comparable.The cTnI concentrations of lithium heparin plasma, EDTA plasma and serum samples were linearly correlated pairwise: EDTA plasma measuremen t =0.76 heparin plasma measurement, R2=0.999(n=40, P<0.001); serum measuremen t =1.05 heparin plasma measurement,R2=0.996(n=40,P<0.001); serum measuremen t=1.38 EDTA plasma measurement, R2=0.993(n=40,P<0.001).The 99th percentiles were 0.030 ng/ml and 0.035 ng/ml on DXI800 and Access2,respectively,from 1 369 apparently healthy Chinese people.cTnI is significantly higher in elder group than in younger group.The 99th percentiles in 18-30 years old group,31-64 years old group,and≥65 years old group are:0.011 ng/ml,0.029 ng/ml,and 0.035 ng/ml respectively for DXI800;0.023 ng/ml,0.034 ng/ml, and 0.045 ng/ml respectively for Access2.cTnI is significantly higher in men than in women.The 99th percentiles in men and women are: 0.034 ng/ml and 0.032 ng/ml respectively for DXI800;0.043 ng/ml and 0.031 ng/ml respectively for Access2.cTnI was measurable in 62%and 87%of healthy subjects on DXI800 and Access2 systems,respectively.Conclusions The analytical performance of AccuTnI+3 assay fulfills the need of clinical use and the criteria of high-sensitive cardiac troponin assay.

Comparative Study Of Dynamic Hip Screw And Trochanteric Nail For Management Of Stable Intertrochanteric Fracture.

Background& objectives : Intertrochanteric fractures of hip are relatively one of the common fractures and it is imposing a huge burden on patients in terms of medical expenses and morbidity .A sliding hip screw (DHS) and trochanteric nail (TN) both are described for fixation of these fractures. The discussion about the selection of ideal implant is controversial in terms of outcomes in various studies. Methods: Ninety patients with intertrochanteric fracture were treated in our hospital from Jan 2009 to Dec 2011. All AO 31-A1 patients who were between 40-80 years old were included to compare Dynamic hip screw and Trochanteric nail in the management of intertrochanteric fractures by analyzing operative time, duration of hospital stay, complications, time taken to union and post operative mobility. 63 patients were enrolled in DHS group and 27 were enrolled in intertrochanteric nail group.Results: Patients treated with DHS had shorter operative time ,less radiological exposure ,easy reduction and fewer intraoperative and postoperative complications .Implant failure and non union was noted in one out of twenty seven patients treated with trochanteric nail group.Interpretation & Conclusions : The analysis of our study supports the use of DHS rather than trochanteric nail for the treatment of stable intertrochanteric fractures in elderly patients.

A prognostic analysis of patients with pathologic complete response after preoperative neoadjuvant chemoradiotherapy for locally advanced rectal cancer / 中华放射肿瘤学杂志

Objective To analyze the clinical factors for pathologic complete response ( pCR) after preoperative neoadjuvant chemoradiotherapy ( neo?CRT) for locally advanced rectal cancer. Methods From 2005 to 2012, 297 patients with locally advanced rectal cancer and complete clinical data were enrolled as subjects. Those patients were diagnosed with biopsy and treated with neo?CRT ( radiotherapy by 3?dimonsional conformal radiotherapy or volumetric?modulated arc therapy) followed by radical surgery. The logistic regression model was used for the multivariate analyses of the correlation of pCR with age, gender, distance between tumor and the anal verge, serum level of carcinoembryonic antigen ( CEA ) before treatment, hemoglobin level before treatment, cT staging, and cN staging. Results In all patients, 78 ( 26?7%) patients had pCR after treatment. The numbers of patients with pCR were 42( 34?4%) in patients with stage T1?T3 disease and 37(21?1%) in patients with stage T4 disease. In the patients with serum CEA levels no higher than 5?33 ng/ml, 55(36?4%) had pCR after treatment, while in the patients with serum CEA levels higher than 5?33 ng/ml, only 24( 16?4%) had pCR. The univariate analysis revealed that age, gender, distance between tumor and the anal verge, anemia before treatment, or cN staging were not related to pCR. The multivariate analysis showed that stage cT1?T3 and a serum CEA level no higher than 5?33 ng/ml before treatment were influencing factors for pCR after neo?CRT for locally advanced rectal cancer ( P=0?031,P=0?000) . Conclusions The clinical staging and the serum CEA level before treatment are influencing factors for pCR after neo?CRT for locally advanced rectal cancer. The serum CEA level before treatment can be considered as a predictor of pCR after neo?CRT for locally advanced rectal cancer.

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicolaM2

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicolaM2

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Epidemiology and Molecular Characterization of Vancomycin-Resistant Enterococcus faecalis / 대한임상미생물학회지

BACKGROUND: Vancomycin-resistant Enterococci (VRE) infections are caused by Enterococcus faecium in about 90% of the cases but can also be caused by Enterococcus faecalis. Thus, this study investigates factors that cause a low isolation rate of vancomycin-resistant E. faecalis (VREfs). To this end, the authors study the clinical traits, resistant gene structure, genomic classification, and molecular characteristics of the virulent factor. METHODS: From January 2001 through September 2011, 17 vanA-containing E. faecalis isolates were collected from hospitalized patients at Ajou University Hospital in Korea. Identification, antimicrobial susceptibility testing, and PCR of van and esp genes were performed. Pulsed-field gel electrophoresis (PFGE) was used for strain typing. PCR and sequencing of the internal regions of Tn1546 were performed for structural analysis of the van gene. RESULTS: Of 4,235 VRE infections, 3,918 (92.5%) were caused by E. faecium, and 95 (2.2%) were caused by E. faecalis. In 67% of VREfs infections, there was a preceding occurrence of E. faecium infection. All isolates were of genotype vanA. Our isolates were divided into three types according to the distribution of IS elements integrated into Tn1546 (types I to IIb). The PFGE results showed no clonal relatedness among isolates. CONCLUSION: Our study found that VREfs infections affect patients who have experienced vancomycin-resistant E. faecium. (VREfm) infection or undergo invasive procedures. The VREfs seems to involve the horizontal transfer of Tn1546 transposon from VREfm.

Effects of 5-azacytidine on radiation-induced epithelial-mesenchymal transition in rat alveolar type Ⅱ epithelial cell line / 中华放射肿瘤学杂志

Objective To investigate the effects of 5-azacytidine on radiation-induced epithelialmesenchymal transition in rat alveolar type Ⅱ epithelial cell line (RLE-6TN) and explore their working mechanisms,and to provide experimental evidence for the potential drug-based treatment of radiationinduced pulmonary fibrosis.Methods RLE-6TN cells were cultured in vitro and divided into four groups according to the experimental purposes:control group (C),radiation group (R),5-azacytidine group (A),and radiation followed by 5-azacytidine group (R + A).The microstructural changes in cells were determined by transmission electron microscopy.Inverted phase-contrast microscopy revealed the morphological changes in cells.The mRNA expression levels of E-cadherin and α-SMA were measured by quantitative real-time polymerase chain reaction (qRT-PCR).The protein expression levels of E-cadherin,GSK3 β,and p-GSK3 β (Ser9) were measured by Western blot.The one-way analysis of variance was used for pairwise comparison.Results The cells in group R became spindle-like.Similar morphological changes were not observed in cells in group R + A.Osmiophilic lamellar bodies disappeared at last in cells in group R.RT-PCR results showed that compared with group C,group R had a significantly lower mRNA expression level of E-cadherin ((0.23 ± 0.06) vs.(1.00 ± 0.00),P =0.002)) and a significantly higher mRNA expression level of α-SMA ((2.91 ± 0.01) vs.(1.00 ± 0.00),P =0.000)).However,compared with group R,group R + A had a significantly higher mRNA expression level of E-cadherin ((0.47 ± 0.05) vs.(1.00 ± 0.00),P =0.024)) but a significantly lower mRNA expression level of α-SMA ((2.50 ± 0.02) vs.(1.00 ±0.00),P =0.037)).The results of Western blot showed that the protein expression level of Ecadherin was significantly reduced ((0.07 ± 0.01) vs.(0.48 ± 0.02),P =0.028)),while the protein expression level of p-GSK3β was significantly increased in Group R than in Group C ((0.85 ± 0.04) vs.(0.23 ± 0.03),P =0.031)).However,compared with group R,group R + A had a significantly lower protein expression level of E-cadherin ((0.25 ± 0.00) vs.(0.07 ± 0.01),P =0.024)) and significantly less up-regulation of the protein expression level of p-GSK3β ((0.39 ± 0.03) vs.(0.85 ± 0.04),P =0.014)).Conclusions X-ray radiation can induce the epithelial-mesenchymal transition in epithelial cells.5-azacytidine suppresses radiation-induced epithelial-mesenchymal transition by inhibition of the activity of p-GSK3β in RLE-6TN cells.

The mechanism of Tn antigen expression in human colon cancer HT-29 cells / 中华微生物学和免疫学杂志

Objective To explore the mechanism of Tn antigen expression in colon cancer cells HT-29.Methods Tn(+) and Tn(-) cells were separated from human colon cancer cell line HT-29 by im-mune magnetic beads.Total RNA, genomic DNA ( gDNA) and cytoplasmic proteins in these cells were ex-tracted by using Trizol, DNA preparation kit and nuclear and cytoplasmic extraction reagents respectively.T-synthase activity was measured by a fluorescent assay.Cosmc and T-synthase transcriptional levels were ana-lyzed by RT-PCR using mRNA as the templates.The coding sequence ( CDS) and CpG islands of Cosmc were amplified by PCR using gDNA as the templates.Amplified products were analyzed on 1%agarose gel. The expected bands were purified, and then sequenced to examine Cosmc mutation.Wild type Cosmc ( Wt-Cosmc) were transfected into tumor cell lines and normal cells to define the function of Cosmc.The expres-sions of Cosmc protein in these cells were then examined by Western blot.Results Although no mutation appeared in HT-29-Tn(-) cells, the deletion of CDS and inactivity of T-synthase were observed in HT-29-Tn(+) cells.Additionally, transfected WtCosmc restored T-synthase activity and then decreased Tn antigen expression in Tn antigen positive cells.Conclusion The absence of CDS in Cosmc gene resulted in the loss of T-synthase activity and consequent Tn antigen expression in HT-29-Tn(+) cells.

Caracterização molecular de Enterobacteriaceae não-Klebsiella pneumoniae produtoras de KPC isoladas em diferentes estados brasileiros / Molecular characterization of non-Enterobacteriaceae Klebsiella pneumoniae KPC-producing isolated in different Brazilian states

A produção de carbapenemases do tipo KPC tem se tornado um importante mecanismo de resistência aos carbapenemas na família Enterobacteriaceae. Embora seja descrita predominantemente em Klebsiella pneumoniae, a enzima KPC também tem sido encontrada em diferentes espécies de Enterobacteriaceae. Contudo, pouco se sabe sobre a epidemiologia da disseminação do gene blaKPC-2 nestas outras espécies. No Brasil, a enzima KPC foi relatada inicialmente em K. pneumoniae no Recife, em 2006, mas atualmente já se encontra disseminada pelo país, onde sua incidência tem aumentado significativamente. Portanto, o objetivo desse trabalho foi realizar a caracterização molecular de amostras brasileiras produtoras de KPC pertencentes a diferentes espécies de Enterobacteriaceae (excluindo K. pneumoniae), isoladas de diferentes estados brasileiros no período de 2009 a 2011. O perfil de resistência foi avaliado por difusão em ágar e E-test. A variante alélica de blaKPC, assim como a participação do transposon Tn4401 e a análise da presença de outros genes de beta-lactamases (TEM, SHV e CTX-M) foram realizadas por PCR e sequenciamento. Análise plasmidial e hibridação foram realizadas para determinar o ambiente genético do gene blaKPC. Para a tipagem molecular foi realizado PFGE e MLST (somente para Escherichia coli)Foram encaminhadas ao Laboratório de Pesquisa em Infecção Hospitalar da Fundação Oswaldo Cruz, 83 amostras produtoras de KPC-2 (correspondendo as espécies: Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Pantoea agglomerans, Providencia stuartii, Citrobacter freundii, Klebsiella oxytoca, Morganella morganii e Serratia marcescens) provenientes de 9 estados das regiões sudeste, nordeste e centro-oeste do país. Em amostras KPC positivas, foram encontrados altos percentuais de resistência à maioria dos antimicrobianos testados, inclusive tigeciclina (36,1 porcento não sensíveis) e polimixina B (16,5 porcento)...

The production of Klebsiella pneumoniae carbapenemase (KPC)-type enzymes has become an important mechanism of carbapenem resistance in the Family Enterobacteriaceae.Although it is predominantly described in Klebsiella pneumoniae, the KPC enzyme has also been found in different species of Enterobacteriaceae. Moreover, little is known about theepidemiology of the dissemination of blaKPC gene in other Enterobacteriaceae species. InBrazil, KPC was initially described in K. pneumoniae in Recife, state of Pernambuco, in 2006, but currently this enzyme is already disseminated throughout the country, where itsincidence has increased significantly. Thus, this study aimed to perform the molecular characterization of KPC-producing brazilian isolates belonging to different species of Enterobacteriaceae (non-K. pneumoniae) originated from different Brazilian states between2009 and 2011. The resistance profile was evaluated by disc-diffusion method and E-test. The allelic variant of the blaKPC gene, as well as the participation of Tn4401 and the presence of other beta-lactamase genes (TEM, SHV and CTX-M) were analyzed by PCR and genome sequencing. Plasmid analysis and hibridization were used to determine the genetic environment of the blaKPC gene. Molecular typing was done by PFGE and MLST (only forEscherichia coli). Eighty three unique clinical isolates of Enterobacteriaceae KPC-2-producers were referred to the Laboratório de Pesquisa em Infecção Hospitalar from Fundação Oswaldo Cruz, corresponding to 9 different species (Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Pantoea agglomerans, Providencia stuartii, Citrobacter freundii, Klebsiella oxytoca, Morganella morganii and Serratia marcescens) isolated from 9 states located in northeast, southeast and central regions of Brazil. Highresistance rates towards most of the antimicrobial agents tested, including tigecycline (36.1 percent nonsusceptible) and polymyxin B (16.5 percent) were detected...

Effects of mutation in Cosmc non-coding region on the transcription level of Cosmc mRNA in Tn antigen positive tumor cells / 中华微生物学和免疫学杂志

Objective To explore the mutation in corel β3-galactosyl-transferase specific molecular chaperone(Cosmc、no-coding region and it's effects on the transcription level of Cosmc in Tn antigen positive tumor cells.Methods The Tn antigen positive(Tn+)and negative(Tn-)cells were separated from tumor tissues by immune magnetic bead,then the genomic DNA(gDNA),total RNA were prepared by Qiagen AllPrep DNA/RNA mini kit. In these cells.the transcription levels of T-synthase and Cosmc mRNA were tested by RT-PCR.the DNA of Cosmc non-coding region was amplified by PCR,the mutation in Cosmc non-coding region were further detected by sequencing.Results There are no mutation appearing in Tn-cells,one or more mosaic sequence allele appearing in portion of patient's Tn-cells.Almost of the Tn+cells which separated from tumor tissues and Jurkat T cell exists mutation.but the mutation style and mutation point were not saine in different tumor.Thtee patient's Tn+cells have loss of hetemzygosity(LOH),four patient's Tn+cells and Jurkat T cell have point mutation.Although no difference of transcription level of T-synthase mRNA in Tn+ and Tn-cells.but the transcription level of Cosmc mRNA in Tn+ cell was much lower than that in Tn-cell.The ratio of T-synthase/Cosmc mRNA in Tn+ tumor cells was hiigher than that in Tn-cell.Conclusion The tumor Tn antigen arise from mutation in Cosmc non-coding region maybe result from transcription level decreased of Cosmc mRNA.

PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.

CHARACTERIZATION OF Salvia bogotensis ANTI-LECTINIGYS AND THEIR APPLICATION IN IMMUNOCYTOCHEMICAL STUDIES INVOLVING TN ANTIGEN DETECTION / CARACTERIZACIÓN DE IGYS ANTI-LECTINA DE Salvia bogotensis Y SU APLICACIÓN EN ESTUDIOS CITOQUÍMICOS PARA LA DETECCIÓN DEL ANTÍGENO TN / CARACTERIZAÇÃO DA IGYS ANTI-LECTINA DE Salvia bogotensis E A SUA APLICAÇÃO EM ESTUDOS CITOQUÍMICOS PARA A DETECÇÃO DO ANTIGÉNIO TN

Immunoglobulins isolated from egg yolk (IgY) are tools which are currently used in different fields of the biological sciences; they have clear advantages over mammalian serum IgGs. We have established the conditions for obtaining anti-Salvia bogotensis lectin IgYs in previous work; their use in immunocytochemical studies requires that their main molecular characteristics are known as well as the conditions for IgY-lectin interaction. Salvia bogotensis lectin (SBoL) can specifically recognise Tn antigen, a recognised tumoral marker in many types of cancer but additional tools are required for evidencing this interaction in cells. Given the availability of S. bogotensis anti-lectin IgY, this work was aimed at molecularly characterising these IgYs and evaluating their application in immunocytochemical studies for detecting Tn antigen in tumour cells. Purified IgYs' isoelectric points, molecular weight and carbohydrate content were determined. Homologous and heterologous lectins were obtained for establishing the specificity of antibody interaction with the lectin; they were assayed by ELLSA. Biotin-or peroxidase-labelled IgYs were prepared; Tn antigen was specifically detected by CELISA and immunocytochemistry in MCF-7 and HeLa cell-lines with the lectin which was revealed with the labelled IgYs. Results showed that anti-SBoL IgY antibodies represent a highly sensitive tool for specific Tn antigen recognition assays.

Las inmunoglobulinas aisladas de la yema de huevo (IgY) son muy utilizadas actualmente en diversos campos de las ciencias biológicas, dadas sus ventajas frente a las IgG séricas de mamíferos. En un trabajo previo establecimos las condiciones de obtención de IgY dirigidas contra la lectina de Salvia bogotensis; su utilización en estudios inmunocitoquímicos requiere conocer sus principales características moleculares y las condiciones para la interacción IgY-lectina. La lectina de Salvia bogotensis (SBoL) reconoce específicamente el antígeno Tn, marcador tumoral en muchos tipos de cáncer, pero se requieren herramientas adicionales para evidenciar esta interacción a nivel celular. Dada la disponibilidad de IgY anti-lectina de S. bogotensis, se realizó este trabajo con el objeto de caracterizar molecularmente estas IgY y evaluar su utilización en estudios inmunocitoquímicos para la detección del antígeno Tn en células tumorales. A las IgY purificadas se les determinó su punto isoeléctrico, peso molecular y contenido de carbohidratos. Para establecer la especificidad de interacción IgY-SBoL se obtuvieron lectinas homólogas y heterólogas y se ensayaron por ELLSA. La detección del antígeno Tn en las líneas celulares MCF-7 y HeLa con la lectina y las IgYs marcadas con biotina o peroxidasa se realizó por CELISA e inmunocitoquímica. Los resultados mostraron que los anticuerpos IgY anti-SBoL son una herramienta de una alta sensibilidad para los ensayos de reconocimiento específico del antígeno Tn.

Imunoglobulinas isoladas a partir de gema de ovo (IgY) são ferramentas utilizadas actualmente em diferentes áreas das ciências biológicas y apresentam vantagens claras sobre o soro de mamífero IgGs. Em investigações anteriores estabelecemos as condições para obter lectina IgYs anti-Salvia bogotensis. O seu uso em estudos imunocitoquímicos requer que as suas principais características moleculares sejam conhecidas, assim como as condições para a interacção IgY-lectina. A lectina de Salvia bogotensis (SBoL) pode reconhecer especificamente o anti-génio Tn, um reconhecido marcador tumoral em muitos tipos de cancro, mas novas ferramentas são necessárias para evidenciar esta interacção em células. Dada a disponibilidade de anti-lectina IgY de S. bogotensis, este trabalho teve como objectivo a caracterização molecular de estes IgYs e avaliar a sua aplicação em estudos imunocitoquímicos para detectar antigénio Tn em células tumorais. Foram determinados os pontos isoeléctricos, peso molecular e conteúdo de carbohidratos de IgYs purificadas. Lectinas homologas e heterologas foram obtidas para estabelecer a especificidade da interacção de anticorpo com a lectina; estes foram ensaiados por ELLSA. Foram preparados IgYs marcados com biotina ou peroxidase. O antigénio Tn foi detectado especificamente por CELISA e imunocitoquimica em linhas celulares MCF-7 y HeLa com lectina que foi revelada com as IgYs marcadas. Os resultados mostraram que os anticorpos anti-SBoL IgY representam uma ferramenta altamente especifica para ensaios de reconhecimento especifico de antigenio Tn.