Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the 'Beijing' sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive

VvNPR1.1 est l’orthologue d’AtNPR1 et sa surexpression provoque l’activation constitutive des gènes PR et la résistance à Erysiphe necator chez Vitis vinifera

La compréhension des bases moléculaires des mécanismes de résistance de la Vigne aux agresseurs biotiques constitue un prérequis à la recherche de moyens de lutte alternatifs aux pesticides. Chez Arabidopsis, NPR1 (Non expressor of PR genes 1) joue un rôle clé dans la voie de signalisation régulée par l’acide salicylique et responsable de la mise en place de la résistance aux agents pathogènes biotrophes et de la résistance systémique acquise (SAR). Nous avons identifié deux gènes homologues d’AtNPR1 chez la Vigne : VvNPR1.1 et VvNPR1.2. La caractérisation fonctionnelle de ces deux gènes montre que la surexpression de VvNPR1.1 dans le mutant npr1-2 d’Arabidopsis permet, contrairement `a VvNPR1.2, de restaurer l’expression de PR1 après traitement par du SA ou inoculation bactérienne, ainsi que la résistance à Pseudomonas syringae pv. maculicola, un agent pathogène virulent. VvNPR1.1 apparaît donc comme l’orthologue fonctionnel d’AtNPR1, alors que VvNPR1.2 assure vraisemblablement une fonction différente. La surexpression stable de VvNPR1.1 en fusion avec la GFP a également pu être réalisée chez V. vinifera cv. Chardonnay, grâce à une technique de transformation par A. tumefaciens de cals embryogènes de Vigne. Les résultats obtenus sur les plantules transformées montrent une localisation constitutive de VvNPR1-GFP dans le noyau, ainsi qu’une expression élevée des protéines PR en l’absence d’infection. De plus, les vignes surexprimant VvNPR1-GFP montrent clairement une augmentation de la r´esistance vis-à-vis de l’infection par Erysiphe necator, l’agent de l’oïdium. La forte conservation de séquence des gènes VvNPR1 chez les Vitaceae ainsi que l’ensemble de ces résultats souligne l’importance de la voie régulée par le SA et NPR1 pour la résistance aux agents pathogènes biotrophes chez la Vigne

Protein-protein interactions in the RPS4/RRS1 immune receptor complex

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation

A Non Mouse-Adapted Dengue Virus Strain as a New Model of Severe Dengue Infection in AG129 Mice

The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing

A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria

Therapeutic targeting of Krüppel-like factor 4 abrogates microglial activation

<p>Abstract</p> <p>Background</p> <p>Neuroinflammation occurs as a result of microglial activation in response to invading micro-organisms or other inflammatory stimuli within the central nervous system. According to our earlier findings, Krüppel-like factor 4 (Klf4), a zinc finger transcription factor, is involved in microglial activation and subsequent release of proinflammatory cytokines, tumor necrosis factor alpha, macrophage chemoattractant protein-1 and interleukin-6 as well as proinflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-treated microglial cells. Our current study focuses on finding the molecular mechanism of the anti-inflammatory activities of honokiol in lipopolysaccharide-treated microglia with emphasis on the regulation of Klf4.</p> <p>Methods</p> <p>For <it>in vitro </it>studies, mouse microglial BV-2 cell lines as well as primary microglia were treated with 500 ng/mL lipopolysaccharide as well as 1 μM and 10 μM of honokiol. We cloned full-length Klf4 cDNA in pcDNA3.1 expression vector and transfected BV-2 cells with this construct using lipofectamine for overexpression studies. For <it>in vivo </it>studies, brain tissues were isolated from BALB/c mice treated with 5 mg/kg body weight of lipopolysaccharide either with or without 2.5 or 5 mg/kg body weight of honokiol. Expression of Klf4, cyclooxygenase-2, inducible nitric oxide synthase and phospho-nuclear factor-kappa B was measured using immunoblotting. We also measured the levels of cytokines, reactive oxygen species and nitric oxide in different conditions.</p> <p>Results</p> <p>Our findings suggest that honokiol can substantially downregulate the production of proinflammatory cytokines and inflammatory enzymes in lipopolysaccharide-stimulated microglia. In addition, honokiol downregulates lipopolysaccharide-induced upregulation of both Klf4 and phospho-nuclear factor-kappa B in these cells. We also found that overexpression of Klf4 in BV-2 cells suppresses the anti-inflammatory action of honokiol.</p> <p>Conclusions</p> <p>Honokiol potentially reduces inflammation in activated microglia in a Klf4-dependent manner.</p

Paracrine cyclooxygenase-2 activity by macrophages drives colorectal adenoma progression in the Apc Min/+ mouse model of intestinal tumorigenesis

Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression in vivo in the ApcMin/+ mouse model of intestinal tumorigenesis. Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal (but not small intestinal) adenomas from cLys-Cox-2 x ApcMin/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control ApcMin/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear β-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of ApcMin/+ mouse colonic adenomas, linked to increased epithelial cell β-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer

A Novel Neural Substrate for the Transformation of Olfactory Inputs into Motor Output

Anatomical and physiological experiments in the lamprey reveal the neural circuit involved in transforming olfactory inputs into motor outputs, which was previously unknown in a vertebrate