Feature extraction and signal processing for nylon DNA microarrays

BACKGROUND: High-density DNA microarrays require automatic feature extraction methodologies and softwares. These can be a potential source of non-reproducibility of gene expression measurements. Variation in feature location or in signal integration methodology may be a significant contribution to the observed variance in gene expression levels. RESULTS: We explore sources of variability in feature extraction from DNA microarrays on Nylon membrane with radioactive detection. We introduce a mathematical model of the signal emission and derive methods for correcting biases such as overshining, saturation or variation in probe amount. We also provide a quality metric which can be used qualitatively to flag weak or untrusted signals or quantitatively to modulate the weight of each experiment or gene in higher level analyses (clustering or discriminant analysis). CONCLUSIONS: Our novel feature extraction methodology, based on a mathematical model of the radioactive emission, reduces variability due to saturation, neighbourhood effects and variable probe amount. Furthermore, we provide a fully automatic feature extraction software, BZScan, which implements the algorithms described in this paper

Olfactory Stem Cells, a New Cellular Model for Studying Molecular Mechanisms Underlying Familial Dysautonomia

International audienceBackground: Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSC) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.Methodology/Principal Findings: We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and identified 2 novel spliced isoforms also present in control cells. We observed a significant lower expression of both IKBKAP transcript and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. We also investigated cellular pathways altered in FD, at the genome-wide level, and confirmed that cell migration and cytoskeleton reorganization were among the processes altered in FD. Indeed, FD hOE-MSCs exhibit impaired migration compared to control cells. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation.Conclusions/Significance: hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases

The transcriptional signatures of Sodalis glossinidius in the Glossina palpalis gambiensis flies negative for Trypanosoma brucei gambiense contrast with those of this symbiont in tsetse flies positive for the parasite : possible involvement of a Sodalis-hosted prophage in fly Trypanosoma refractoriness ?

Tsetse flies, such as Glossina palpalis gambiensis, are blood-feeding insects that could be subverted as hosts of the parasite Trypanosoma brucei gambiense: initiated in the tsetse fly mid gut, the developmental program of this parasite further proceeds in the salivary glands. The flies act as vectors of this human-invasive parasite when their salivary glands sustain the generation of metacyclic trypomastigotes, the exclusive morphotypes pre-programmed to further develop in the human individuals. Briefly, once the metacyclic trypomastigotes have been deposited in the skin of humans from whom the parasite-hosting tsetse flies are taking their blood meals, the complex developmental program of this Trypanosoma brucei subspecies can result in a severe disease named sleeping sickness. Unveiling the processes that could prevent, in tsetse flies, the developmental program of T. b. gambiense could contribute reducing the prevalence of the disease. Using a global approach, we were curious to extract transcriptional signatures of Sodalis glossinidius, a symbiont hosted by three distinct groups of tsetse flies. To meet this objective, the transcriptome of S. glossinidius from susceptible and refractory tsetse flies was analyzed at 3, 10 and 20 days after flies blood feed on T. b. gambiense-hosting mice. Within this temporal window, 176 trypanosome responsive genes were shown to interact in well-defined patterns making it possible to distinguish flies susceptible to the parasite infection from refractory flies. Among the modulated transcripts in the symbiont population of flies refractory to trypanosome infection many were shown to cluster within the following networks: "lysozyme activity, bacteriolytic enzyme, bacterial cytolysis, cell wall macromolecule catabolic process". These novel data are further delineated in the following questions: could the activation of prophage hosted by S. glossinidius lead to the release of bacterial agonists that trigger the tsetse fly immune system along a profile that no more allows the parasite developmental program? (C) 2014 Elsevier B.V. All rights reserved

A general survey of thymocyte differentiation by transcriptional analysis of knockout mouse models

International audienceThe thymus is the primary site of T cell lymphopoiesis. To undergo proper differentiation, developing T cells follow a well-ordered genetic program that strictly depends on the heterogeneous and highly specialized thymic microenvironment. In this study, we used microarray technology to extensively describe transcriptional events regulating alphabeta T cell fate. To get an integrated view of these processes, both whole thymi from genetically engineered mice together with purified thymocytes were analyzed. Using mice exhibiting various transcriptional perturbations and developmental blockades, we performed a transcriptional microdissection of the organ. Multiple signatures covering both cortical and medullary stroma as well as various thymocyte maturation intermediates were clearly defined. Beyond the definition of histological and functional signatures (proliferation, rearrangement), we provide the first evidence that such an approach may also highlight the complex cross-talk events that occur between maturing T cells and stroma. Our data constitute a useful integrated resource describing the main gene networks set up during thymocyte development and a first step toward a more systematic transcriptional analysis of genetically modified mice

Genome-wide analysis of familial dysautonomia and kinetin target genes with patient olfactory ecto-mesenchymal stem cells.

International audienceFamilial dysautonomia (FD) is a rare inherited neurodegenerative disorder. The most common mutation is a c.2204+6T>C transition in the 5' splice site (5'ss) of IKBKAP intron 20, which causes a tissue-specific skipping of exon 20, resulting in lower synthesis of IKAP/hELP1 protein. To better understand the specificity of neuron loss in FD, we modeled the molecular mechanisms of IKBKAP mRNA splicing by studying human olfactory ecto-mesenchymal stem cells (hOE-MSCs) derived from FD patient nasal biopsies. We explored how the modulation of IKBKAP mRNA alternative splicing impacts the transcriptome at the genome-wide level. We found that the FD transcriptional signature was highly associated with biological functions related to the development of the nervous system. In addition, we identified target genes of kinetin, a plant cytokinin that corrects IKBKAP mRNA splicing and increases the expression of IKAP/hELP1. We identified this compound as a putative regulator of splicing factors and added new evidence for a sequence-specific correction of splicing. In conclusion, hOE-MSCs isolated from FD patients represent a promising avenue for modeling the altered genetic expression of FD, demonstrating a methodology that can be applied to a host of other genetic disorders to test the therapeutic potential of candidate molecules

Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2

International audienceThrombin exerts pleiotropic effects on endothelial cells, including the release of microparticles (EMPs) that disseminate and exchange information with vascular cells. Nevertheless, the mechanisms leading to their generation are not elucidated. We performed microarray analysis to identify genes involved in EMP release by the endothelial cell line HMEC-1 in response to thrombin. We identified a group of genes linked to the cytoskeleton reorganization family. Among these, the Rho-kinase ROCK-II presented a high transcription rate. ROCK-I, another Rho-kinase isoform, was not modulated by thrombin. Pharmacologic inhibition of Rho-kinases or specific depletion of ROCK-II by short interfering (si) RNA inhibited thrombin-induced EMP release. In contrast, ROCK-I mRNA silencing did not modify EMP generation by thrombin. Exposure of HMEC-1 to thrombin in presence of the caspase-2 selective inhibitor Z-VDVAD-FMK prevented ROCK-II cleavage and inhibited the thrombin-induced EMP release. These events were observed in absence of cell death. Our data clearly identified ROCK-II as a target of thrombin in EMP generation. They indicated that the 2 Rho-kinases did not share identical functions. The involvement of caspase-2 in ROCK-II activation independently of cell death points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in EMP generation in response to cell activation

ORIGINAL IN-SILICO APPROACH IDENTIFIES NEW BROAD-SPECTRUM INFLUENZA A ANTIVIRALS

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Gene expression profiling during thymus ontogeny and its association with TCRV beta 8.1-D beta 2.1 rearrangements of inbred mouse strains

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