The GENCODE pseudogene resource.

BACKGROUND: Pseudogenes have long been considered as nonfunctional genomic sequences. However, recent evidence suggests that many of them might have some form of biological activity, and the possibility of functionality has increased interest in their accurate annotation and integration with functional genomics data. RESULTS: As part of the GENCODE annotation of the human genome, we present the first genome-wide pseudogene assignment for protein-coding genes, based on both large-scale manual annotation and in silico pipelines. A key aspect of this coupled approach is that it allows us to identify pseudogenes in an unbiased fashion as well as untangle complex events through manual evaluation. We integrate the pseudogene annotations with the extensive ENCODE functional genomics information. In particular, we determine the expression level, transcription-factor and RNA polymerase II binding, and chromatin marks associated with each pseudogene. Based on their distribution, we develop simple statistical models for each type of activity, which we validate with large-scale RT-PCR-Seq experiments. Finally, we compare our pseudogenes with conservation and variation data from primate alignments and the 1000 Genomes project, producing lists of pseudogenes potentially under selection. CONCLUSIONS: At one extreme, some pseudogenes possess conventional characteristics of functionality; these may represent genes that have recently died. On the other hand, we find interesting patterns of partial activity, which may suggest that dead genes are being resurrected as functioning non-coding RNAs. The activity data of each pseudogene are stored in an associated resource, psiDR, which will be useful for the initial identification of potentially functional pseudogenes.

Roles of transient guidepost neurons in corpus callosum formation and guidance

RESUMENeurones transitoires jouant un rôle de cibles intermédiaires dans le guidage des axones du corps calleuxLe guidage axonal est une étape clé permettant aux neurones d'établir des connexions synaptiques et de s'intégrer dans un réseau neural fonctionnel de manière spécifique. Des cellules-cibles intermédiaires appelées « guidepost » aident les axones à parcourir de longues distances dans le cerveau en leur fournissant des informations directionnelles tout au long de leur trajet. Il a été démontré que des sous-populations de cellules gliales au niveau de la ligne médiane guident les axones du corps calleux (CC) d'un hémisphère vers l'autre. Bien qu'il fût observé que le CC en développement contenait aussi des neurones, leur rôle était resté jusqu'alors inconnu.La publication de nos résultats a montré que pendant le développement embryonnaire, le CC contient des glies mais aussi un nombre considérable de neurones glutamatergiques et GABAergiques, nécessaires à la formation du corps calleux (Niquille et al., PLoS Biology, 2009). Dans ce travail, j'ai utilisé des techniques de morphologie et d'imagerie confocale 3D pour définir le cadre neuro-anatomique de notre modèle. De plus, à l'aide de transplantations sur tranches in vitro, de co-explants, d'expression de siRNA dans des cultures de neurones primaires et d'analyse in vivo sur des souris knock-out, nous avons démontré que les neurones du CC guident les axones callosaux en partie grâce à l'action attractive du facteur de guidage Sema3C sur son récepteur Npn- 1.Récemment, nous avons étudié l'origine, les aspects dynamiques de ces processus, ainsi que les mécanismes moléculaires impliqués dans la mise en place de ce faisceau axonal (Niquille et al., soumis). Tout d'abord, nous avons précisé l'origine et l'identité des neurones guidepost GABAergiques du CC par une étude approfondie de traçage génétique in vivo. J'ai identifié, dans le CC, deux populations distinctes de neurones GABAergiques venant des éminences ganglionnaires médiane (MGE) et caudale (CGE). J'ai ensuite étudié plus en détail les interactions dynamiques entre neurones et axones du corps calleux par microscopie confocale en temps réel. Puis nous avons défini le rôle de chaque sous-population neuronale dans le guidage des axones callosaux et de manière intéressante les neurones GABAergic dérivés de la MGE comme ceux de la CGE se sont révélés avoir une action attractive pour les axones callosaux dans des expériences de transplantation. Enfin, nous avons clarifié la base moléculaire de ces mécanismes de guidage par FACS sorting associé à un large criblage génétique de molécules d'intérêt par une technique très sensible de RT-PCR et ensuite ces résultats ont été validés par hybridation in situ.Nous avons également étudié si les neurones guidepost du CC étaient impliqués dans son agénésie (absence de CC), présente dans nombreux syndromes congénitaux chez 1 humain. Le gène homéotique Aristaless (Arx) contrôle la migration des neurones GABAergiques et sa mutation conduit à de nombreuses pathologies humaines, notamment la lissencéphalie liée à IX avec organes génitaux anormaux (XLAG) et agénésie du CC. Fait intéressant, nous avons constaté qu'ARX est exprimé dans toutes les populations GABAergiques guidepost du CC et que les embryons mutant pour Arx présentent une perte drastique de ces neurones accompagnée de défauts de navigation des axones (Niquille et al., en préparation). En outre, nous avons découvert que les souris déficientes pour le facteur de transcription ciliogenic RFX3 souffrent d'une agénésie du CC associé avec des défauts de mise en place de la ligne médiane et une désorganisation secondaire des neurones glutamatergiques guidepost (Benadiba et al., submitted). Ceci suggère fortement l'implication potentielle des deux types de neurones guidepost dans l'agénésie du CC chez l'humain.Ainsi, mon travail de thèse révèle de nouvelles fonctions pour ces neurones transitoires dans le guidage axonal et apporte de nouvelles perspectives sur les rôles respectifs des cellules neuronales et gliales dans ce processus.ABSTRACTRole of transient guidepost neurons in corpus callosum development and guidanceAxonal guidance is a key step that allows neurons to build specific synaptic connections and to specifically integrate in a functional neural network. Intermediate targets or guidepost cells act as critical elements that help to guide axons through long distance in the brain and provide information all along their travel. Subpopulations of midline glial cells have been shown to guide corpus callosum (CC) axons to the contralateral cerebral hemisphere. While neuronal cells are also present in the developing corpus callosum, their role still remains elusive.Our published results unravelled that, during embryonic development, the CC is populated in addition to astroglia by numerous glutamatergic and GABAergic guidepost neurons that are essential for the correct midline crossing of callosal axons (Niquille et al., PLoS Biology, 2009). In this work, I have combined morphological and 3D confocal imaging techniques to define the neuro- anatomical frame of our system. Moreover, with the use of in vitro transplantations in slices, co- explant experiments, siRNA manipulations on primary neuronal culture and in vivo analysis of knock-out mice we have been able to demonstrate that CC neurons direct callosal axon outgrowth, in part through the attractive action of Sema3C on its Npn-1 receptor.Recently, we have studied the origin, the dynamic aspects of these processes as well as the molecular mechanisms involved in the establishment of this axonal tract (Niquille et al., submitted). First, we have clarified the origin and the identity of the CC GABAergic guidepost neurons using extensive in vivo cell fate-mapping experiments. We identified two distinct GABAergic neuronal subpopulations, originating from the medial (MGE) and caudal (CGE) ganglionic eminences. I then studied in more details the dynamic interactions between CC neurons and callosal axons by confocal time-lapse video microscopy and I have also further characterized the role of each guidepost neuronal subpopulation in callosal guidance. Interestingly, MGE- and CGE-derived GABAergic neurons are both attractive for callosal axons in transplantation experiments. Finally, we have dissected the molecular basis of these guidance mechanisms by using FACS sorting combined with an extensive genetic screen for molecules of interest by a sensitive RT-PCR technique, as well as, in situ hybridization.I have also investigated whether CC guidepost neurons are involved in agenesis of the CC which occurs in numerous human congenital syndromes. Aristaless-related homeobox gene (Arx) regulates GABAergic neuron migration and its mutation leads to numerous human pathologies including X-linked lissencephaly with abnormal genitalia (XLAG) and severe CC agenesis. Interestingly, I found that ARX is expressed in all the guidepost GABAergic neuronal populations of the CC and that Arx-/- embryos exhibit a drastic loss of CC GABAergic interneurons accompanied by callosal axon navigation defects (Niquille et al, in preparation). In addition, we discovered that mice deficient for the ciliogenic transcription factor RFX3 suffer from CC agenesis associated with early midline patterning defects and a secondary disorganisation of guidepost glutamatergic neurons (Benadiba et al., submitted). This strongly points out the potential implication of both types of guidepost neurons in human CC agenesis.Taken together, my thesis work reveals novel functions for transient neurons in axonal guidance and brings new perspectives on the respective roles of neuronal and glial cells in these processes.

Bone marrow mesenchymal stem cells stabilize already-formed aortic aneurysms more efficiently than vascular smooth muscle cells in a rat model.

PURPOSE: Abdominal aortic aneurysms (AAAs) expand because of aortic wall destruction. Enrichment in Vascular Smooth Muscle Cells (VSMCs) stabilizes expanding AAAs in rats. Mesenchymal Stem Cells (MSCs) can differentiate into VSMCs. We have tested the hypothesis that bone marrow-derived MSCs (BM-MSCs) stabilizes AAAs in a rat model. MATERIAL AND METHODS: Rat Fischer 344 BM-MSCs were isolated by plastic adhesion and seeded endovascularly in experimental AAAs using xenograft obtained from guinea pig. Culture medium without cells was used as control group. The main criteria was the variation of the aortic diameter at one week and four weeks. We evaluated the impact of cells seeding on inflammatory response by immunohistochemistry combined with RT-PCR on MMP9 and TIMP1 at one week. We evaluated the healing process by immunohistochemistry at 4 weeks. RESULTS: The endovascular seeding of BM-MSCs decreased AAA diameter expansion more powerfully than VSMCs or culture medium infusion (6.5% ± 9.7, 25.5% ± 17.2 and 53.4% ± 14.4; p = .007, respectively). This result was sustained at 4 weeks. BM-MSCs decreased expression of MMP-9 and infiltration by macrophages (4.7 ± 2.3 vs. 14.6 ± 6.4 mm(2) respectively; p = .015), increased Tissue Inhibitor Metallo Proteinase-1 (TIMP-1), compared to culture medium infusion. BM-MSCs induced formation of a neo-aortic tissue rich in SM-alpha active positive cells (22.2 ± 2.7 vs. 115.6 ± 30.4 cells/surface units, p = .007) surrounded by a dense collagen and elastin network covered by luminal endothelial cells. CONCLUSIONS: We have shown in this rat model of AAA that BM-MSCs exert a specialized function in arterial regeneration that transcends that of mature mesenchymal cells. Our observation identifies a population of cells easy to isolate and to expand for therapeutic interventions based on catheter-driven cell therapy.

Adipose reduction in the activity of the repressor ICER is responsible for insulin resistance elicited by the transcriptional factors CREB in obesity

BACKGROUND AND AIMS: Sustained adipose activation of the transcriptional activators cAMP response binding proteins (CREB) in obesity leads to impaired expression of the glucose transporter GLUT4 and adiponectin (adipoq) in mice model of obesity. Diminution of GLUT4 and adipoq caused by CREB is indirect and relies on the increased repressive activity of the CREB target gene activating transcription factor 3 (ATF3). Specific inactivation of CREB in adipocytes decreases ATF3 production and improves whole-body insulin sensitivity of mice in the context of diet-induced obesity. Thus, elevation of CREB activity is a key mechanism responsible for adipocyte dysfunction and systemic insulin resistance. The inducible cAMP early repressor (ICER) is a negative regulator of the CREB activity. In fact, ICER antagonizes the CREB factor by competing for the regulation of similar target genes. The goal of the study was to investigate whether loss of ICER expression in adipocytes could be responsible for increased CREB activity in obesity. MATERIALS AND METHODS: Mice C57bl6 were fed with a high fat diet (HFD) for 12 weeks to increase body weight and generate insulin resistance. Biopsies of visceral adipose tissues (VAT) were prepared from human lean (BMI=24}0.5 Kg/m2) or obese subjects (BMI>35 Kg/m2). Total RNA and protein were prepared from white adipose tissues (WAT) of chow- or HFD-fed mice and VAT of lean and obese subjects. Activities of CREBs and ICER were monitored by electromobility shift assays (EMSA). The role of ICER on CREB activity was confirmed in 3T3-L1 adipocytes cells. Briefly after differentiation, the cells were electroporated with the plasmid coding for ICER cDNA. Gene expression was quantified by quantitative real-time PCR and western Blotting experiments. RESULTS: The expression of ICER is reduced in WAT of HFD-induced obese mice when compared to chow mice as measured by real-time PCR and EMSA. Similar result was found in human tissues. Reduction in ICER expression was associated with increased ATF3 expression and decreased adipoq and GLUT4 contents. Diminution in ICER levels was observed in adipocytes fraction whereas its expression was unchanged in stroma vascular fraction of WAT. Overexpression of ICER in 3T3-L1 adipocytes silenced the expression of ATF3, confirming the regulation of the factor by ICER. The expression of ICER is regulated by histone deacetylases activity (HDAC). Inhibition of HDACs in 3T3-L1 adipocytes cells using trichostatin inhibited the production of ICER. The whole activity of HDAC was reduced in WAT and VAT of obese mice and human obese subjects. CONCLUSION: Impaired adipose expression of ICER is responsible of increased CREB activity in adipocytes in obesity. This mechanism relies on reduction of the HDAC activity.

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia.

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Evaluation of peanut genotypes for resistance to Tomato spotted wilt virus by mechanical and thrips inoculation

The objective of this work was to evaluate the reactions of three peanut breeding lines (IC-10, IC-34, and ICGV 86388) to Tomato spotted wilt virus (TSWV) by mechanical and thrips inoculation, under greenhouse conditions, and compare them to the reactions of cultivars SunOleic, Georgia Green, and the breeding line C11-2-39. TSWV infection by mechanical inoculation was visually assessed using an index ranging from 0 (no symptoms) to 4 (apical death). Enzyme-linked immunosorbent assay was used to confirm TSWV infection from both mechanical and thrips inoculations. IC-10, IC-34, ICGV 86388, and C11-2-39 were more resistant than the cultivars SunOleic and Georgia Green based on mechanical inoculation. Upon thrips inoculation only IC-34 and ICGV-86388 were infected by TSWV, as demonstrated by reverse transcription polymerase chain reaction (RT-PCR), although no symptoms of infection were observed. The peanut breeding lines IC-10, IC-34, and ICGV 86388 show higher level of resistance to TSWV than cultivar Georgia Green considered a standard for TSWV resistance.

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Iontophoresis of dexamethasone in the treatment of endotoxin-induced-uveitis in rats.

The purpose of this study was to evaluate the efficacy of a Coulomb Controlled Iontophoresis system (CCI) in the local delivery of corticosteroids for the treatment of uveitis. The therapeutic efficacy of Dexamethasone (Dex) administered by CCI was compared to systemic injection and to topical application with the iontophoresis apparatus in the absence of electrical current. The evaluation was done in the treatment of the endotoxin-induced uveitis (EIU) model, and in the effect on TNF gene expression in the iris/ciliary body as well as in the retina and on TNF levels in aqueous humor and vitreous. Dex was administered either at the time of LPS injection or 5 hours later. For iontophoresis, we used a 1 ml reservoir-electrode covering the cornea, the limbus, and the first millimeter of the sclera. The applied electrical current was of 400 microA during four minutes with a total surface charge of 0.4 C cm-2. EIU was evaluated by clinical examination, by counts of intraocular inflammatory cells on histological sections, and by measuring the protein levels in the aqueous humor and in the vitreous. The TNF-alpha gene expression in the iris and ciliary body, and in the retina was evaluated by RT-PCR. The systemic effect of Dex delivered by CCI was evaluated on the level of serum TNF-alpha in EIU. Our results demonstrated that local administration of Dex by CCI inhibited anterior and posterior signs of intraocular inflammation as effectively as systemic administration, with no effect on systemic level of TNF. In the anterior and posterior segments of the eye, the protein exudation. TNF levels and the cellular infiltration were inhibited. The TNF-alpha gene expression was inhibited in the anterior as well as the posterior segment of the eye. No clinical nor histological damage were caused by the CCI apparatus. In conclusion, CCI administration of Dex allows for a therapeutic effect on the posterior as well as the anterior segment of the eye, and may present a viable alternative to systemic administration of glucocorticoids in severe ocular inflammations.

Characterization of BMP signaling in breast development and tumorigenesis

Résumé L'influence des hormones reproductives sur le développement du cancer du sein a été établie au travers de nombreuse études épidémiologiques. Nous avons précédemment démontré que le gène Wnt-4 est un médiateur essentiel de la progestérone dans le développement lobulo-alvéolaire de l'épithélium mammaire. De plus, le rôle de la voie de signalisation Wnt dans la tumorigénèse de la glande mammaire mutine est largement établi. Pour comprendre sa fonction dans le cancer du sein, nous avons activée cette voie en surexprimant le gène Wnt-1 dans des cellules épithéliales primaires de sein, au moyen d'un rétrovirus. Ceci a conduit à la transformation oncogénique de ces cellules et à l'obtention d'un modèle de carcinogénèse du sein dénommé Wnt-1 HMEC. L'analyse de l'expression des gènes induits par la surexpression de Wnt-1 dans ces cellules, a permis d'identifier les gènes BMP4 et 7. Alors que des analyses de RT-PCR ont montré leur forte expression dans les cellules Wnt-1-HMECs, la présence d'une grande quantité de la protéine BMP7 a été constatée dans les tumeurs dérivées de ces cellules. L'importante phosphorylation des Smad 1, 5, S dans les Wnt-1 HMECs indique l'activation de la voie BMP, possiblement due à la stimulation ce celle-ci par BMP7. L'activation de la voie Wnt par la ß-Caténine, conduit à la transcription de BMP7, identifiant ainsi ce gène comme un gène cible de la voie canonique. La pertinence de nos observations a par ailleurs été confirmée par le fait que BMP7 est surexprimé dans les tumeurs de seins humains. Afin d'élucider la fonction de la voie BMP dans le sein, nous avons utilisé le modèle mutin. L'expression du gène BMP7 dans les souris transgéniques MMTV Wnt-1 s'est avérée élevée, démontrant qu'il est aussi un gène cible de la voie Wnt in-vivo. L'expression de l'ARN messager .codant pour la protéine BMP7 est induite lors du développement lobulo-alvéolaire, qui se fait sous l'influence de la progestérone et de Wnt-4. Ensemble, ces observations corroborent le fait qu'une stimulation avec de la progestérone suffit à induire la transcription du gène dans les 24h. Nos résultats coïncident d'autre part avec le fait que BMP7 est exprimé dans la couche myoépithéliale de l'épithélium où la voie Wnt est activée. L'analyse de souris reportrices de l'activité de la voie BMP, suggère une activation dans la couche luminale de l'épithélium durant tout le développement de la glande mammaire. Curieusement, cette même voie est active dans le mésenchyme lors de la mammogénèse embryonnaire. Finalement, nos analyses d'immunofluorescence démontrent la capacité de prolifération des cellules ayant activé BMP, ainsi que leur nette ségrégation d'avec les cellules exprimant le récepteur à la progestérone. Nos résultats démontrent que le gène BMP7 est un gène cible de la voie Wnt canonique dans le sein. Son expression dans la couche myoépitheliale est induite par Wnt-4, lui-même sécrété par les cellules luminales sensibles à la progestérone. La sécrétion de la protéine BMP7 conduit finalement à l'activation de la voie BMP dans les cellules négatives pour le récepteur à la progestérone. Abstract Epidemiological studies highlight the repetitive exposure to circulating progesterone as a major risk in the development of breast cancer. Work in our laboratory showed that Wnt-4 is an essential mediator of progesterone-driven side-branch formation, while Wnt signaling has long been established as strongly oncogenic in the mouse mammary gland. To address the role of Wnt in breast tumorigenesis we activated the pathway in primary human breast epithelial cells by means of refroviral Wnt-1 expression. This resulted in a Wnt1-induced breast carcinogenesis model, being referred to as Wnt-1-HMECs. Gene expression profiling revealed the Bone Morphogenetic Protein 4 and 7 (BMP4 and 7) a mong the most upregulated gene by ectopic Wnt-1 expression in primary HMECs. RT-PCR analysis confirmed elevated BMP4 and 7 mRNA levels in Wnt-1-infected HMECs, as well as strong BMP7 expression in the tumors derived from these cells. Smad 1, 5, 8 phosphorylation was high in Wnt-1HMECs whereas below detection limit in primary HMECs suggesting that the increased expression of BMP-7 results in activation of downstream signaling. Ectopic expressíon of a stabilized form of ßcatenin in primary HMECs resulted in increased transcription of BMP-7 suggesting that it is a target of canonical Wnt signaling. The clinical relevance of our observations was confirmed by the finding of BMP7 being upregulated in human breast tumor samples. To elucidate the role of BMP ligands in the breast in-vivo, we made use of the mouse model. Expression of the BMP7 gene was found to be increased in MMTV-Wnt-1 transgenic animals, suggesting that BMP7 may also be a Wnt 1 target gene in vivo. Expression of BMP7 was upregulated in mid-pregnancy which coincides with progesterone/Wnt induced side branching. BMP7 was induced within 24 hours by progesterone. Consistent with it being a target of canonical Wnt signaling, we demonstrated preferential expression of this ligand in the myoepithelial cells, the target cells of Wnt signals. In-vivo analysis of BMP signaling using a reporter mouse revealed the activation of the pathway in the luminal layer of the epithelium throughout postnatal development. Interestingly, during embryonic mammogenesis the pathway was found to be active in the mesenchyme. Immunofluorescence studies demonstrated that cells with BMP activity can proliferate. They also revealed a clear segregation between progesterone receptor positive cells and cells with active BMP signaling. Together our observations suggest that BMP-7 is a canonical Wnt signaling target both in HMECs and in the mouse mammary gland in-vivo. It is expressed in the myoepithelium possibly in response to Wnt-4, which is secreted by steroid receptor positive cells in response to progesterone. BMP-7 in turn may impinge on lumina) epithelial cells and activate BMP signaling in PR negative cells.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Characterization of a new potential virulence factor of Microsporum canis, the secreted subtilisin Sub6

Background: Microsporum canis is a dermatophyte responsible for cutaneous superficial mycoses in domestic carnivores and humans. The pathogenesis of dermatophytoses, including M. canis infections, remains poorly understood. Secreted proteases including members of the subtilisin family are thought to be involved in the infection process. In particular the subtilisin Sub6 could represent a major virulence factor.Objective: The aim of this work was to (i) isolate the M. canis SUB6 genomic DNA and cDNA (ii) produce Sub6 as a recombinant protease (rSub6) and (iii) produce a specific anti-Sub6 polyclonal serum. Material and methods: Genomic SUB6 was amplified by PCR using specific primers and M. canis IHEM 21239 DNA as a target. The SUB6 cDNA was obtained by reverse transcriptase (RT)-PCR using total RNA extracted from the same M. canis strain grown in liquid medium containing feline keratin as unique nitrogen source. Both SUB6 cDNA and genomic DNA were sequenced. The SUB6 cDNA was cloned in pPICZA to produce recombinant Sub6 (rSub6) in Pichia pastoris KM71. This protease rSub6 was produced in methanol medium at a yield of 30 mg ml)1 and purified by anion exchange chromatography using a DEAE-sepharose column. Polyclonal antibodies against purified rSub6 were produced in a rabbit using a standard immunization procedure with saponin as the adjuvant. Seventy days after the first immunization, serum was collected and IgG were purified by affinity chromatography.Results: The coding sequence for M. canis SUB6 from genomic DNA contains 1410 bp and 3 introns, while the cDNA contains a 1221 bp open reading frame. Deduced amino acid sequence analysis revealed that Sub6 is synthesized as a 406 amino acids preproprotein. The predicted catalytic domain has 286 amino acids, a molecular mass of 29.1 kDa and five potential N-glycosylation sites. SDS-PAGE of rSub6 revealed a single polypeptide chain with an apparent molecular mass of 37 kDa. Purified rabbit IgG were shown to be specific for Sub6 using ELISA.Conclusion: We have characterized for the first time Sub6 from a dermatophyte species as a recombinant secreted active enzyme and purified it until homogeneity. Active rSub6 and Sub6 specific antiserum will be used to further study the role of M. canis Sub6 protease in pathogenesis, notably the pattern of in vivo Sub6 secretion in different host species.

Epidemiology and new developments in the diagnosis of prosthetic joint infection.

Although prosthetic joint infection (PJI) is a rare event after arthroplasty, it represents a significant complication that is associated with high morbidity, need for complex treatment, and substantial healthcare costs. An accurate and rapid diagnosis of PJI is crucial for treatment success. Current diagnostic methods in PJI are insufficient with 10-30% false-negative cultures. Consequently, there is a need for research and development into new methods aimed at improving diagnostic accuracy and speed of detection. In this article, we review available conventional diagnostic methods for the diagnosis of PJI (laboratory markers, histopathology, synovial fluid and periprosthetic tissue cultures), new diagnostic methods (sonication of implants, specific and multiplex PCR, mass spectrometry) and innovative techniques under development (new laboratory markers, microcalorimetry, electrical method, reverse transcription [RT]-PCR, fluorescence in situ hybridization [FISH], biofilm microscopy, microarray identification, and serological tests). The results of highly sensitive diagnostic techniques with unknown specificity should be interpreted with caution. The organism identified by a new method may represent a real pathogen that was unrecognized by conventional diagnostic methods or contamination during specimen sampling, transportation, or processing. For accurate interpretation, additional studies are needed, which would evaluate the long-term outcome (usually >2 years) with or without antimicrobial treatment. It is expected that new rapid, accurate, and fully automatic diagnostic tests will be developed soon.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients. RESULTS: Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages. CONCLUSIONS: CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.