Advanced spot quality analysis in two-colour microarray experiments

Background: Image analysis of microarrays and, in particular, spot quantification and spot quality control, is one of the most important steps in statistical analysis of microarray data. Recent methods of spot quality control are still in early age of development, often leading to underestimation of true positive microarray features and, consequently, to loss of important biological information. Therefore, improving and standardizing the statistical approaches of spot quality control are essential to facilitate the overall analysis of microarray data and subsequent extraction of biological information. Findings: We evaluated the performance of two image analysis packages MAIA and GenePix (GP) using two complementary experimental approaches with a focus on the statistical analysis of spot quality factors. First, we developed control microarrays with a priori known fluorescence ratios to verify the accuracy and precision of the ratio estimation of signal intensities. Next, we developed advanced semi-automatic protocols of spot quality evaluation in MAIA and GP and compared their performance with available facilities of spot quantitative filtering in GP. We evaluated these algorithms for standardised spot quality analysis in a whole-genome microarray experiment assessing well-characterised transcriptional modifications induced by the transcription regulator SNAI1. Using a set of RT-PCR or qRT-PCR validated microarray data, we found that the semi-automatic protocol of spot quality control we developed with MAIA allowed recovering approximately 13% more spots and 38% more differentially expressed genes (at FDR = 5%) than GP with default spot filtering conditions. Conclusion: Careful control of spot quality characteristics with advanced spot quality evaluation can significantly increase the amount of confident and accurate data resulting in more meaningful biological conclusions. © 2008 Friederich et al; licensee BioMed Central Ltd

Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model

Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1–LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family

Design and evaluation of Actichip, a thematic microarray for the study of the actin cytoskeleton

<p>Abstract</p> <p>Background</p> <p>The actin cytoskeleton plays a crucial role in supporting and regulating numerous cellular processes. Mutations or alterations in the expression levels affecting the actin cytoskeleton system or related regulatory mechanisms are often associated with complex diseases such as cancer. Understanding how qualitative or quantitative changes in expression of the set of actin cytoskeleton genes are integrated to control actin dynamics and organisation is currently a challenge and should provide insights in identifying potential targets for drug discovery. Here we report the development of a dedicated microarray, the Actichip, containing 60-mer oligonucleotide probes for 327 genes selected for transcriptome analysis of the human actin cytoskeleton.</p> <p>Results</p> <p>Genomic data and sequence analysis features were retrieved from GenBank and stored in an integrative database called Actinome. From these data, probes were designed using a home-made program (CADO4MI) allowing sequence refinement and improved probe specificity by combining the complementary information recovered from the UniGene and RefSeq databases. Actichip performance was analysed by hybridisation with RNAs extracted from epithelial MCF-7 cells and human skeletal muscle. Using thoroughly standardised procedures, we obtained microarray images with excellent quality resulting in high data reproducibility. Actichip displayed a large dynamic range extending over three logs with a limit of sensitivity between one and ten copies of transcript per cell. The array allowed accurate detection of small changes in gene expression and reliable classification of samples based on the expression profiles of tissue-specific genes. When compared to two other oligonucleotide microarray platforms, Actichip showed similar sensitivity and concordant expression ratios. Moreover, Actichip was able to discriminate the highly similar actin isoforms whereas the two other platforms did not.</p> <p>Conclusion</p> <p>Our data demonstrate that Actichip is a powerful alternative to commercial high density microarrays for cytoskeleton gene profiling in normal or pathological samples. Actichip is available upon request.</p

A Novel Network Integrating a miRNA-203/SNAI1 Feedback Loop which Regulates Epithelial to Mesenchymal Transition

BACKGROUND: The majority of human cancer deaths are caused by metastasis. The metastatic dissemination is initiated by the breakdown of epithelial cell homeostasis. During this phenomenon, referred to as epithelial to mesenchymal transition (EMT), cells change their genetic and trancriptomic program leading to phenotypic and functional alterations. The challenge of understanding this dynamic process resides in unraveling regulatory networks involving master transcription factors (e.g. SNAI1/2, ZEB1/2 and TWIST1) and microRNAs. Here we investigated microRNAs regulated by SNAI1 and their potential role in the regulatory networks underlying epithelial plasticity. RESULTS: By a large-scale analysis on epithelial plasticity, we highlighted miR-203 and its molecular link with SNAI1 and the miR-200 family, key regulators of epithelial homeostasis. During SNAI1-induced EMT in MCF7 breast cancer cells, miR-203 and miR-200 family members were repressed in a timely correlated manner. Importantly, miR-203 repressed endogenous SNAI1, forming a double negative miR203/SNAI1 feedback loop. We integrated this novel miR203/SNAI1 with the known miR200/ZEB feedback loops to construct an a priori EMT core network. Dynamic simulations revealed stable epithelial and mesenchymal states, and underscored the crucial role of the miR203/SNAI1 feedback loop in state transitions underlying epithelial plasticity. CONCLUSION: By combining computational biology and experimental approaches, we propose a novel EMT core network integrating two fundamental negative feedback loops, miR203/SNAI1 and miR200/ZEB. Altogether our analysis implies that this novel EMT core network could function as a switch controlling epithelial cell plasticity during differentiation and cancer progression

LSST: from Science Drivers to Reference Design and Anticipated Data Products

(Abridged) We describe here the most ambitious survey currently planned in the optical, the Large Synoptic Survey Telescope (LSST). A vast array of science will be enabled by a single wide-deep-fast sky survey, and LSST will have unique survey capability in the faint time domain. The LSST design is driven by four main science themes: probing dark energy and dark matter, taking an inventory of the Solar System, exploring the transient optical sky, and mapping the Milky Way. LSST will be a wide-field ground-based system sited at Cerro Pach\'{o}n in northern Chile. The telescope will have an 8.4 m (6.5 m effective) primary mirror, a 9.6 deg$^2$ field of view, and a 3.2 Gigapixel camera. The standard observing sequence will consist of pairs of 15-second exposures in a given field, with two such visits in each pointing in a given night. With these repeats, the LSST system is capable of imaging about 10,000 square degrees of sky in a single filter in three nights. The typical 5$\sigma$ point-source depth in a single visit in $r$ will be $\sim 24.5$ (AB). The project is in the construction phase and will begin regular survey operations by 2022. The survey area will be contained within 30,000 deg$^2$ with $\delta<+34.5^\circ$, and will be imaged multiple times in six bands, $ugrizy$, covering the wavelength range 320--1050 nm. About 90\% of the observing time will be devoted to a deep-wide-fast survey mode which will uniformly observe a 18,000 deg$^2$ region about 800 times (summed over all six bands) during the anticipated 10 years of operations, and yield a coadded map to $r\sim27.5$. The remaining 10\% of the observing time will be allocated to projects such as a Very Deep and Fast time domain survey. The goal is to make LSST data products, including a relational database of about 32 trillion observations of 40 billion objects, available to the public and scientists around the world.Comment: 57 pages, 32 color figures, version with high-resolution figures available from https://www.lsst.org/overvie

Caractérisation du mécanisme de transport nucléo-cytoplasmique de la protéine P25 et mise en évidence d'interactions entre les protéines de mouvement du virus des nervures jaunes et nécrotiques de la betterave

Le BNYVV est responsable de la rhizomanie de la betterave sucrière. Son génome est consitué de 4 à 5 RNA de polarité positive. Les résultats décrits dans ce mémoire concernent, d'une part, l'étude du transport nucléo-cytoplasmique de la protéine P25 codée par le RNA3 ainsi que son influence sur la symptomatologie, et d'autre part, la localisation subcellulaire et l'étude des interactions entre les trois protéines virales impliquées dans le mouvement à courte distance du virus. Des études antérieures ont montré que la P25 est responsable des symptômes typiques de la rhizomanie. Grâce à des observations en microscopie confocale, j'ai montré que la P25 fusionnée à la GFP, se localise dans le cytoplasme et dans le noyau des cellules infectées. J'ai mis alors en évidence sur la P25, un signal de localisation nucléaire (NLS) ainsi qu'un signal d'exportation nucléaire (NEMS). Le transport de la P25 du cytoplasme vers le noyau fait intervenir une interaction directe entre la séquence NLS et l'importine a. L'exportation nucléaire, quant à elle, se fait grâce à la séquence NEMS par la voie des Exportines1. Des expériences de mutagénèse dirigée sur des sites potentiels de phosphorylation semblent montrer que la régulation du transport nucléo-cytoplasmique fait intervenir des protéines kinases. Des études sur la symptomatologie foliaire et la localisation subcellulaire des P25, sauvage ou mutées, exprimées en contexte viral, montrent que les formes mutées de la P25, incapables de transiter dans le noyau, n'induisent plus les symptômes associés à l'expression de la P25. Enfin, des expériences de "simple hybride" ont révélé que la P25 était capable d'activer la transcription de gènes rapporteurs de levure. L'ensemble de ces résultats, nous laisse penser que la P25, qui contient en outre un motif " doigt à zinc " et un domaine C-terminal acide, pourrait moduler la transcription de gènes cellulaires, ce qui expliquerait les symptômes caractéristiques de la rhizomanie. Dans une deuxième partie, j'ai montré, par microscopie électronique que les protéines de mouvement TGBp1 et TGBp2 colocalisaient au niveau des plasmodesmes de cellules infectées par le BNYVV. L'existence d'interactions entre ces deux protéines a été confirmée par des expériences de farwestern et de co-immunoprécipitation.Beet necrotic yellow vein virus (BNYVV) is responsible for rhizomania disease of sugar beet. Its genome is composed of 4 or 5 plus-sense RNAs. The results described in this thesis concern (1) the nucleo-cytoplasmic transport of the protein P25 coded by BNYVV RNA 3 and its influence on symptomatology, and (2) the subcellular localization and the interactions among the three viral proteins implicated in cell-to-cell movement of the virus. Previous studies showed that P25 is responsible for the typical symptoms of rhizomania. By means of laser scanning confocal microscopy, I have shown that P25 fused to GFP localizes to both the cytoplasm and the nucleus of infected cells. Both a nuclear localization signal (NLS) and a nuclear export signal (NEMS) were detected on P25. The transport of P25 from the cytoplasm to the nucleus involved a direct interaction between the NLS and importin-a. Nuclear export required the NEMS sequence and occured by the Exportin1 pathway. Mutagenesis of potential phosphorylation sites on P25 indicated that its nuclear-cytoplasmic trafficking was regulated by protein kinases. A correlative study of leaf symptoms and the subcellular localization of wild-type and mutant P25's expressed in the context of a viral infection showed that nuclear import-deficient P25 mutants did not induce the leaf symptoms associated with P25 expression. Finally, one-hybrid experiments revealed that P25 can activate transcription of reporter genes in yeast. Taken together, these results suggest that P25, which contains a Zn-finger motif and a C-terminal acidic domain, could modulate transcription of cellular genes, and this could explain the characteristic symptoms of rhizomania. In the second part of my thesis, I have used electron microscopy to show that the BNYVV movement proteins TGBp1 and TGBp2 co-localize at plasmodesmata of BNYVV-infected cells. The existence of interactions between the two proteins was confirmed by farwestern and co-immunoprecipitation experiments.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Involvement of the Secretory Pathway and the Cytoskeleton in Intracellular Targeting and Tubule Assembly of Grapevine fanleaf virus Movement Protein in Tobacco BY-2 Cells

Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein–MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized

Estrogen and retinoic acid antagonistically regulate several microRNA genes to control aerobic glycolysis in breast cancer cells.

International audienceIn addition to estrogen receptor modulators, retinoic acid and other retinoids are promising agents to prevent breast cancer. Retinoic acid and estrogen exert antagonistic regulations on the transcription of coding genes and we evaluated here whether these two compounds have similar effects on microRNAs. Using an integrative approach based on several bioinformatics resources together with experimental validations, we indeed found that retinoic acid positively regulates miR-210 and miR-23a/24-2 expressions and is counteracted by estrogen. Conversely, estrogen increased miR-17/92 and miR-424/450b expressions and is inhibited by retinoic acid. In silico functional enrichment further revealed that this combination of transcriptional/post-transcriptional regulations fully impacts on the molecular effects of estrogen and retinoic acid. Besides, we unveiled a novel effect of retinoic acid on aerobic glycolysis. We specifically showed that it increases extracellular lactate production, an effect counteracted by the miR-210 and the miR-23a/24-2, which simultaneously target lactate dehydrogenase A and B mRNAs. Together our results provide a new framework to better understand the estrogen/retinoic acid antagonism in breast cancer cells