Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappaB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC DeltanleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappaB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappaB response elements, we found that NleH causes an increase in NF-kappaB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.

CYR61 and TAZ Upregulation and Focal Epithelial to Mesenchymal Transition May Be Early Predictors of Barrett’s Esophagus Malignant Progression

Barrett's esophagus is the major risk factor for esophageal adenocarcinoma. It has a low but non-neglectable risk, high surveillance costs and no reliable risk stratification markers. We sought to identify early biomarkers, predictive of Barrett's malignant progression, using a meta-analysis approach on gene expression data. This in silico strategy was followed by experimental validation in a cohort of patients with extended follow up from the Instituto Português de Oncologia de Lisboa de Francisco Gentil EPE (Portugal). Bioinformatics and systems biology approaches singled out two candidate predictive markers for Barrett's progression, CYR61 and TAZ. Although previously implicated in other malignancies and in epithelial-to-mesenchymal transition phenotypes, our experimental validation shows for the first time that CYR61 and TAZ have the potential to be predictive biomarkers for cancer progression. Experimental validation by reverse transcriptase quantitative PCR and immunohistochemistry confirmed the up-regulation of both genes in Barrett's samples associated with high-grade dysplasia/adenocarcinoma. In our cohort CYR61 and TAZ up-regulation ranged from one to ten years prior to progression to adenocarcinoma in Barrett's esophagus index samples. Finally, we found that CYR61 and TAZ over-expression is correlated with early focal signs of epithelial to mesenchymal transition. Our results highlight both CYR61 and TAZ genes as potential predictive biomarkers for stratification of the risk for development of adenocarcinoma and suggest a potential mechanistic route for Barrett's esophagus neoplastic progression.

Criblage génétique à la recherche de nouveaux gènes essentiels influençant l’homéostasie des télomères chez Saccharomyces cerevisiae : Un défi de tailles.

Résumé : Chez la levure Saccharomyces cerevisiae, la régulation de la longueur des télomères témoigne de la compensation entre mécanismes d'érosion (exonucléases, réplication semi-conservative et résection), facteurs d’élongation (la télomérase, transcriptase inverse à l'action retrouvée dans 90% des cancers humains) et actions de diverses protéines de régulation télomérique spécifiques, conférant aux télomères leur caractère de « capuchon » protégeant les extrémités des chromosomes eucaryotes. Afin de savoir si les gènes impossibles à déléter, car essentiels à la survie cellulaire, jouent aussi un rôle sur l’homéostasie télomérique, j'ai réalisé un criblage génétique utilisant des mutants tet-off de la levure pour lesquels la sous-expression considérable d'un gène essentiel a été induite de façon conditionnelle. Ceci permet d’étudier les effets qui en résultent sur l’homéostasie des télomères. Au total, mon travail a traité plus de 662 gènes essentiels pour lesquels j'ai analysé le phénotype de longueur des télomères de manière qualitative par comparaison des télomères de souches mutées par rapport à ceux de souches de type sauvage. Puis, grâce à l’amélioration technique que j'ai mise au point, la quantification de la taille des répétitions télomériques de 300 de ces souches a déjà pu être précisément analysée. Il est notable que tous les gènes essentiels étudiés ici ont des effets très différents qui résultent en des chromosomes possédant des télomères de longueur très inégale. Pour près de 40% des mutants analysés, les tailles de télomères sont apparues critiquement différentes de celles normalement présentées par la levure, beaucoup de ces gènes essentiels étant impliqués dans des mécanismes affectant le cycle cellulaire, la réparation, etc. La majorité des gènes criblés apporte un important complément d’information dans une littérature presque inexistante sur les effets de gènes essentiels de la levure au niveau de la biologie des télomères. C’est le cas des mutations de YHR122W (montrant des télomères long) et YOR262W (télomères courts), deux gènes qui sont apparus d'après mes résultats nécessaires au maintien de l'homéostasie télomérique (prenant place dans un grand ensemble de gènes que j’ai dénommé gènes ETL pour Essential for Telomere Length Maintenance).

Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients

International audience

Exogenous Administration of 15d-PGJ(2)-Loaded Nanocapsules Inhibits Bone Resorption in a Mouse Periodontitis Model

The 15-deoxy-(Delta 12,14)-PG J(2) (15d-PGJ(2)) has demonstrated excellent anti-inflammatory results in different experimental models. It can be used with a polymeric nanostructure system for modified drug release, which can change the therapeutic properties of the active principle, leading to increased stability and slower/prolonged release. The aim of the current study was to test a nano-technological formulation as a carrier for 15d-PGJ(2), and to investigate the immunomodulatory effects of this formulation in a mouse periodontitis model. Poly (D, L-lactide-coglycolide) nanocapsules (NC) were used to encapsulate 15d-PGJ(2). BALB/c mice were infected on days 0, 2, and 4 with Aggregatibacter actinomycetemcomitans and divided into groups (n = 5) that were treated daily during 15 d with 1, 3, or 10 mu g/kg 15d-PGJ(2)-NC. The animals were sacrificed, the submandibular lymph nodes were removed for FACS analysis, and the jaws were analyzed for bone resorption by morphometry. Immunoinflammatory markers in the gingival tissue were analyzed by reverse transcriptase-quantitative PCR, Western blotting, or ELISA. Infected animals treated with the 15d-PGJ(2)-NC presented lower bone resorption than infected animals without treatment (p < 0.05). Furthermore, infected animals treated with 10 mu g/kg 15d-PGJ(2)-NC had a reduction of CD4(+)CD25(+)FOXP3(+) cells and CD4/CD8 ratio in the submandibular lymph node (p < 0.05). Moreover, CD55 was upregulated, whereas RANKL was downregulated in the gingival tissue of the 10 mu g/kg treated group (p < 0.05). Several proinflammatory cytokines were decreased in the group treated with 10 mu g/kg 15d-PGJ(2)-NC, and high amounts of 15d-PGJ(2) were observed in the gingiva. In conclusion, the 15d-PGJ(2)-NC formulation presented immunomodulatory effects, decreasing bone resorption and inflammatory responses in a periodontitis mouse model. The Journal of Immunology, 2012, 189: 1043-1052.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Sub-epidemics explain localized high prevalence of reduced susceptibility to Rilpivirine in treatment-naive HIV-1-infected patients: subtype and geographic compartmentalization of baseline resistance mutations

This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Predicted residual activity of rilpivirine in HIV-1 infected patients failing therapy including NNRTIs efavirenz or nevirapine

This is an open access article under the CC BY-NC-ND license - http://creativecommons.org/licenses/by-nc-nd/4.0/

Apoptotic properties of Citrus sudachi Hort, ex Shirai (Rutaceae) extract on humanA549 and HepG2 cancer cells

Purpose: To investigate whether Citrus sudachi harvested at two stages of maturity can induce toxicity in a cell-specific manner and to determine the possible mechanisms of Citrus sudachi-induced cytotoxic responses in two types of cancer cells (human lung adenocarcinoma A549 and hepatocellular carcinoma HepG2 cells) and two normal cell lines (lung 16HBE140- and liver CHANG cells). Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and annexin V/propidium iodidle assay were used to test the antiproliferative activity and apoptosis of methanol extract of Citrus sudachi, respectively. Griess reaction and reverse transcriptase-polymerase chain reaction (RT-PCR) were carried out to evaluate nitric oxide (NO•) production and the mRNA levels of inhibitors of apoptosis (IAP). Results: Citrus sudachi exerted cytotoxicity in a time-dependent manner in cancer cells which increased with increase in maturity but did not affect normal cells. Citrus sudachi was found to induce accumulation of cells in the sub-G1 cell cycle phase, fragmentation of DNA and cell death with characteristics of apoptosis, in both types of cancer cells. Moreover, Citrus sudachi upregulated cellular NO• produced by activation of nitric oxide synthase (NOS), while it suppressed the levels of IAP mRNA in both types of cancer cells. Conclusion: The results obtained suggest that Citrus sudachi induces apoptosis in A549 and HepG2 cells, which may be mediated by NO•. There is need for further studies on the role of Citrus sudachi in cancer treatment.

The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. Objective: This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment.

Transcriptional profiling in Saccharomyces cerevisiae relevant for predicting alachlor mechanisms of toxicity

Alachlor has been a commonly applied herbicide and is a substance of ecotoxicological concern. The present study aims to identify molecular biomarkers in the eukaryotic model Saccharomyces cerevisiae that can be used to predict potential cytotoxic effects of alachlor, while providing new mechanistic clues with possible relevance for experimentally less accessible eukaryotes. It focuses on genome-wide expression profiling in a yeast population in response to two exposure scenarios exerting effects from slight to moderate magnitude at phenotypic level. In particular, 100 and 264 genes, respectively, were found as differentially expressed on a 2-h exposure of yeast cells to the lowest observed effect concentration (110 mg/L) and the 20% inhibitory concentration (200 mg/L) of alachlor, in comparison with cells not exposed to the herbicide. The datasets of alachlor-responsive genes showed functional enrichment in diverse metabolic, transmembrane transport, cell defense, and detoxification categories. In general, the modifications in transcript levels of selected candidate biomarkers, assessed by quantitative reverse transcriptase polymerase chain reaction, confirmed the microarray data and varied consistently with the growth inhibitory effects of alachlor. Approximately 16% of the proteins encoded by alachlor-differentially expressed genes were found to share significant homology with proteins from ecologically relevant eukaryotic species. The biological relevance of these results is discussed in relation to new insights into the potential adverse effects of alachlor in health of organisms from ecosystems, particularly in worst-case situations such as accidental spills or careless storage, usage, and disposal.

Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the “L”-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the “L”-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Culturable Bacterial Diversity From A Feed Water Of A Reverse Osmosis System, Evaluation Of Biofilm Formation And Biocontrol Using Phages.

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.