Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells

Background: In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle. Results: Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-γ), assayed under same conditions. This suggested that proteins other than intimin-γ that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential. Conclusion: Proteins other than LEE and intimin-γ proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines

A gestão escolar como um instrumento de política de línguas

This article analyzes school management as an instrument of language policy. To this end, a discussion of a theoretical nature will first be made, establishing the conditions under which the management of a school can be conceived as an instrument of language policy, with political effects on the way in which the languages in operation in its space are signified and signify its speakers. The school will be conceived as a space of enunciation. In a second moment, an analytical discussion will be made, in which two texts that function as language policy documents at the Federal Institute of São Paulo (IFSP) will be analyzed. The theoretical-analytical discussion will be conducted from the perspective of the semantics of the event, in a fruitful dialog with the French discourse analysis. From the discussion undertaken, it will be observed that the school administration exercises power in the interplay of forces between languages and their speakers in the school space, since, through its administrative-normative acts, it organizes the way each language will function in its space of enunciation.Este artículo analiza la gestión escolar como instrumento de política de lenguas. Para ello, se llevará a cabo una discusión teórica en la que se establecerán las condiciones en las que la gestión de una escuela puede concebirse como un instrumento de política de lenguas, con efectos políticos sobre el modo en que las lenguas que operan en su espacio son significadas y significan a sus hablantes. La escuela será concebida como un espacio de enunciación. En un segundo momento, se realizará una discusión analítica, en la que se analizarán dos textos que funcionan como documentos de política de lenguas en el Instituto Federal de São Paulo (IFSP). La discusión teórico-analítica se realizará desde la perspectiva de la semántica del acontecimiento, en un diálogo fructífero con el análisis del discurso francés. A partir de la discusión emprendida, se observará que la administración escolar ejerce poder en el juego de fuerzas entre las lenguas y sus hablantes en el espacio escolar, ya que, a través de sus actos administrativo-normativos, organiza la forma en que cada lengua funcionará en su espacio de enunciación.Este artigo analisa a gestão escolar como um instrumento de política de línguas. Para isso, far-se-á, primeiramente, uma discussão de natureza teórica, estabelecendo as condições em que a gestão de uma escola pode ser concebida enquanto um instrumento de política de línguas, com efeitos políticos sobre o modo como as línguas em funcionamento em seu espaço são significadas e significam seus falantes. A escola será concebida como um espaço de enunciação. Num segundo momento, será feita uma discussão analítica, na qual serão analisados dois textos que funcionam como documentos de política de línguas no Instituto Federal de São Paulo (IFSP). A discussão teórico-analítica será conduzida sob a perspectiva da semântica do acontecimento, num diálogo profícuo com a análise de discurso de orientação francesa. A partir da discussão empreendida, observar-se-á que a administração escolar exerce poder no jogo de forças entre as línguas e seus falantes no espaço da escola, uma vez que, por meio de seus atos administrativo-normativos, ela organiza o modo como cada língua funcionará em seu espaço de enunciação

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis

Introduction: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression. Methods: Multiplexed high throughput selected reaction monitoring (SRM) assays were developed to measure tryptic peptides representative of 23 proteins in matched serum and synovial fluid samples from late OA subjects at the time of joint replacement. Subsequently plasma samples from the baseline visit of 173 subjects in an observational OA cohort were tested by SRM for peptides from nine of these proteins: afamin, clusterin, cartilage oligomeric matrix protein, hepatocyte growth factor, kallistatin, insulin-like growth factor binding protein, acid labile subunit, lubricin, lumican, and pigment epithelium-derived factor. Linear regression was used to determine the association between the peptide biomarker level at baseline and change in joint space width (ΔJSW) from baseline to 30 months, adjusting for age and sex. Results: In the matched cohort, 17 proteins could be identified in synovial fluid and 16 proteins were detected in serum. For the progression cohort, the average age was 62 and average ΔJSW over 30 months was 0.68 mm. A high correlation between different peptides from individual proteins was observed, indicating our assays correctly measured their target proteins. Peptides representative of clusterin, lumican and lubricin showed statistically significant associations with joint space narrowing after adjustment for age and sex. Partial R2 values showed clusterin FMETVAEK and lubricin LVEVNPK peptide biomarkers explains about 2 to 3% of the variability of ΔJSW, similar to that explained by age. A biomarker score combining normalized data for both lubricin and clusterin peptides increased the model R2 to 0.079. Conclusions: Our results suggest that when combined, levels of peptides representative of clusterin and lubricin in plasma are as predictive of OA progression as age. Replication of these findings in other prospective OA cohorts is planned. Electronic supplementary material The online version of this article (doi:10.1186/s13075-014-0456-6) contains supplementary material, which is available to authorized users

High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis

The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response

La política lingüística de la Universidad Autónoma de Baja California [México] y las estancias cortas virtuales: Una experiencia en la enseñanza del idioma inglés con propósitos específicos

En este estudio se describe una experiencia en la enseñanza del idioma Inglés que realiza la Universidad Autónoma de Baja California (UABC) mediante el programa de intercambio estudiantil virtual en universidades internacionales. Desde 2006, la UABC ha fomentado el aprendizaje del Inglés con el fin de mejorar las competencias lingüísticas y académicas de los universitarios. Una estrategia que se ha empleado para mejorar los programas de Inglés consiste en indagar la opinión de los alumnos que han participado en estancias virtuales cortas en las que se ha utilizado el Inglés como medio de instrucción. Para este estudio se utilizó una metodología cuantitativa-descriptiva para analizar los resultados de 139 alumnos que respondieron la encuesta de 20 ítems con la escala de Likert y validada con Alfa de Cronbach (Cronbach, 1951). El estudio se centra en el referente lingüístico del interpretativismo del aprendizaje del idioma Inglés con Propósitos Específicos (Widdowson, 1998) y las estancias cortas virtuales como parte de la política lingüística explícita de Baldauf (2010). A partir de los resultados obtenidos de este estudio, se emiten recomendaciones para mejorar los cursos de Inglés que se implementan en la UABC para así consolidar la política lingüística establecida por la universidad.&nbsp

The EBNA3 Family of Epstein-Barr Virus Nuclear Proteins Associates with the USP46/USP12 Deubiquitination Complexes to Regulate Lymphoblastoid Cell Line Growth

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation

EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall

Comparative Proteomic Analysis of the PhoP Regulon in Salmonella enterica Serovar Typhi Versus Typhimurium

Background: S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. Methodology/Principal Findings: Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of $phoP^−/Q^−$ mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. Conclusions/Significance: This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800)

Interferon-γ and Proliferation Responses to Salmonella enterica Serotype Typhi Proteins in Patients with S. Typhi Bacteremia in Dhaka, Bangladesh

Salmonella enterica serotype Typhi infection is a significant global public health problem and the cause of typhoid fever. Salmonella are intracellular pathogens, and cellular immune responses are required to control and clear Salmonella infections. Despite this, there are limited data on cellular immune responses during wild type S. Typhi infection in humans. Here we report the assessment of cellular immune responses in humans with S. Typhi bacteremia through a screening approach that permitted us to evaluate interferon-γ and proliferation responses to a number of S. Typhi antigens. We detected significant interferon-γ CD4 and CD8 responses, as well as proliferative responses, to a number of recombinantly purified S. Typhi proteins as well as membrane preparation in infected patients. Antigen-specific interferon-γ responses were present at the time of clinical presentation in patients and absent in healthy controls. These observations could assist in the development of interferon-γ-based diagnostic assays for typhoid fever

Passive cation permeability of turtle colon: Evidence for a negative interaction between intracellular sodium and apical sodium permeability

The role of intracellular sodium in the regulation of apical sodium permeability was investigated in an electrically “tight” epithelium, the turtle colon. In the presence of low mucosal sodium (3 mM) and serosal ouabain, an inhibitor of the basolateral sodium pump, the apical membrane retained a substantial amiloride-sensitive, sodium conductance and the basolateral membrane exhibited a barium-sensitive potassium conductance in parallel with a significant sodium (and lithium) conductance. In the presence of a high mucosal sodium concentration (114 mM), however, inhibition of active sodium absorption by ouabain led to a disappearance of the amiloride-sensitive, transepithelial conductance that was due, at least in part, to a virtual abolition of the apical sodium permeability. Two lines of evidence indicate that this permeability decrease was dependent upon an increase in intracellular sodium content. First, raising the mucosal sodium concentration from 3–114 mM in the presence of ouabain reversibly inhibited the amiloride-sensitive conductance. The time course of the decline in conductance paralleled the apparent intracellular accumulation of sodium in exchange for potassium, which was monitored as a transient deflection in the amiloride-sensitive, short-circuit current. Second, the inhibitory effect of mucosal sodium-addition was markedly attenuated by serosal barium, which prevented the accumulation of sodium by blocking the electrically coupled, basolateral potassium exit. These results support the notion of a “negative feedback” effect of intracellular sodium on the apical sodium permeability.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47451/1/424_2004_Article_BF00583286.pd