The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii

The role of Stl1p in glycerol accumulation in osmotically stressed icewine yeast Saccharomyces cerevisiae K1 V1116

The high sugar concentration in Icewine juice exerts hyperosmotic stress in the wine yeast causing water loss and cell shrinkage. To counteract the dehydration, yeast synthesize and accumulate glycerol as an internal osmolyte. In a laboratory strain of S. cerevisiae, STLl encodes for Stllp, an H+ /glycerol symporter that is glucose inactivated, but induced upon hyperosmotic stress. STLl, was found to be a highly upregulated gene in Icewine fermenting cells and its expression was 25-fold greater than in yeast cells fermenting diluted Icewine juice, making it one of the most differentially expressed genes between the two fermentation conditions. In addition, Icewine fermenting cells showed a two-fold higher glycerol production in the wine compared to yeast fermenting diluted Icewine juice. We proposed that Stllp is (1) active during Icewine fermentation and is not glucose inactivated and (2) its activity contributes to the limited cell growth observed during Icewine fermentation as a result of the dissipation of the plasma membrane proton gradient. To measure the contribution ofStl1p in active glycerol transport (energy dependent) during Icewine fermentation, we first developed an Stllp-dependent (14C]glycerol uptake assay using a laboratory strain of S. cerevisiae (BY 4742 and LiSTLl) that was dependent on the plasma membrane proton gradient and therefore energy-dependent. Wine yeast K1-Vll16 was also shown to have this energy dependent glycerol uptake induced under salt stress. The expression of STLl and Stllp activity were compared between yeast cells harvested from Icewine and diluted Icewine fermentations. Northern blot analysis revealed that STLl was expressed in cells fermenting Icewine juice but not expressed under the diluted juice conditions. Glycerol uptake by cells fermenting Icewine juice was not significantly different than cells fermenting diluted Icewine juice on day 4 and day 7 of Vidal and Riesling fermentations respectively, despite encountering greater hyperosmotic stress. Furthermore, energy- dependent glycerol uptake was not detected under either fermentation conditions. Because our findings show that active glycerol uptake was not detected in yeast cells harvested from Icewine fermentation, it is likely that Stllp was glucose inactivated despite the hyperosmotic stress induced by the Icewine juice and therefore did not play a role in active glycerol uptake during Icewine fermentation.

Probe-level linear model fitting and mixture modeling results in high accuracy detection of differential gene expression

Affiliation: Institut de recherche en immunologie et en cancérologie, Université de Montréal

La régulation génique chez Solanum chacoense : de la pollinisation jusqu’à l’embryogenèse

Chez les végétaux supérieurs, l’embryogenèse est une phase clé du développement au cours de laquelle l’embryon établit les principales structures qui formeront la future plante et synthétise et accumule des réserves définissant le rendement et la qualité nutritionnelle des graines. Ainsi, la compréhension des évènements moléculaires et physiologiques menant à la formation de la graine représente un intérêt agronomique majeur. Toutefois, l'analyse des premiers stades de développement est souvent difficile parce que l'embryon est petit et intégré à l'intérieur du tissu maternel. Solanum chacoense qui présente des fleurs relativement grande facilitant l’isolation des ovules, a été utilisée pour l’étude de la biologie de la reproduction plus précisément la formation des gamètes femelles, la pollinisation, la fécondation et le développement des embryons. Afin d'analyser le programme transcriptionnel induit au cours de la structuration de ces étapes de la reproduction sexuée, nous avons mis à profit un projet de séquençage de 7741 ESTs (6700 unigènes) exprimés dans l’ovule à différents stades du développement embryonnaire. L’ADN de ces ESTs a été utilisé pour la fabrication de biopuces d’ADN. Dans un premier temps, ces biopuces ont été utilisé pour comparer des ADNc issus des ovules de chaque stade de développement embryonnaire (depuis le zygote jusqu’au embryon mature) versus un ovule non fécondé. Trois profils d’expression correspondant au stade précoce, intermédiaire et tardive ont été trouvés. Une analyse plus approfondie entre chaque point étudié (de 0 à 22 jours après pollinisation), a permis d'identifier des gènes spécifiques caractérisant des phases de transition spécifiques. Les annotations Fonctionnelles des gènes differentiellement exprimés nous ont permis d'identifier les principales fonctions cellulaires impliquées à chaque stade de développement, révélant que les embryons sont engagés dans des actifs processus de différenciation. Ces biopuces d’ADN ont été par la suite utilisé pour comparer différent types de pollinisation (compatible, incompatible, semi-compatible et inter-espèce) afin d’identifier les gènes répondants à plusieurs stimuli avant l'arrivé du tube pollinique aux ovules (activation à distance). Nous avons pu démontrer que le signal perçu par l’ovaire était différent et dépend de plusieurs facteurs, incluant le type de pollen et la distance parcourue par le pollen dans le style. Une autre analyse permettant la comparaison des différentes pollinisations et la blessure du style nous a permis d’identifier que les programmes génétiques de la pollinisation chevauchent en partie avec ceux du stress. Cela était confirmé en traitant les fleurs par une hormone de stress, méthyle jasmonate. Dans le dernier chapitre, nous avons utilisé ces biopuces pour étudier le changement transcriptionnel d’un mutant sur exprimant une protéine kinase FRK2 impliqué dans l’identité des ovules. Nous avons pu sélectionner plusieurs gènes candidat touchés par la surexpression de cette kinase pour mieux comprendre la voie se signalisation. Ces biopuces ont ainsi servi à déterminer la variation au niveau transcriptionnelle des gènes impliqués lors de différents stades de la reproduction sexuée chez les plantes et nous a permis de mieux comprendre ces étapes.

Étude des gènes d'Actinobacillus pleuropneumoniae exprimés en conditions d'infection

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes.

Comparaison des méthodes d'analyse de l'expression différentielle basée sur la dépendance des niveaux d'expression

La technologie des microarrays demeure à ce jour un outil important pour la mesure de l'expression génique. Au-delà de la technologie elle-même, l'analyse des données provenant des microarrays constitue un problème statistique complexe, ce qui explique la myriade de méthodes proposées pour le pré-traitement et en particulier, l'analyse de l'expression différentielle. Toutefois, l'absence de données de calibration ou de méthodologie de comparaison appropriée a empêché l'émergence d'un consensus quant aux méthodes d'analyse optimales. En conséquence, la décision de l'analyste de choisir telle méthode plutôt qu'une autre se fera la plupart du temps de façon subjective, en se basant par exemple sur la facilité d'utilisation, l'accès au logiciel ou la popularité. Ce mémoire présente une approche nouvelle au problème de la comparaison des méthodes d'analyse de l'expression différentielle. Plus de 800 pipelines d'analyse sont appliqués à plus d'une centaine d'expériences sur deux plateformes Affymetrix différentes. La performance de chacun des pipelines est évaluée en calculant le niveau moyen de co-régulation par l'entremise de scores d'enrichissements pour différentes collections de signatures moléculaires. L'approche comparative proposée repose donc sur un ensemble varié de données biologiques pertinentes, ne confond pas la reproductibilité avec l'exactitude et peut facilement être appliquée à de nouvelles méthodes. Parmi les méthodes testées, la supériorité de la sommarisation FARMS et de la statistique de l'expression différentielle TREAT est sans équivoque. De plus, les résultats obtenus quant à la statistique d'expression différentielle corroborent les conclusions d'autres études récentes à propos de l'importance de prendre en compte la grandeur du changement en plus de sa significativité statistique.

Transcriptional approach to study porcine tracheal epithelial cells individually or dually infected with swine influenza virus and Streptococcus suis

Background: Swine influenza is a highly contagious viral infection in pigs affecting the respiratory tract that can have significant economic impacts. Streptococcus suis serotype 2 is one of the most important post-weaning bacterial pathogens in swine causing different infections, including pneumonia. Both pathogens are important contributors to the porcine respiratory disease complex. Outbreaks of swine influenza virus with a significant level of co-infections due to S. suis have lately been reported. In order to analyze, for the first time, the transcriptional host response of swine tracheal epithelial (NPTr) cells to H1N1 swine influenza virus (swH1N1) infection, S. suis serotype 2 infection and a dual infection, we carried out a comprehensive gene expression profiling using a microarray approach. Results: Gene clustering showed that the swH1N1 and swH1N1/S. suis infections modified the expression of genes in a similar manner. Additionally, infection of NPTr cells by S. suis alone resulted in fewer differentially expressed genes compared to mock-infected cells. However, some important genes coding for inflammatory mediators such as chemokines, interleukins, cell adhesion molecules, and eicosanoids were significantly upregulated in the presence of both pathogens compared to infection with each pathogen individually. This synergy may be the consequence, at least in part, of an increased bacterial adhesion/invasion of epithelial cells previously infected by swH1N1, as recently reported. Conclusion: Influenza virus would replicate in the respiratory epithelium and induce an inflammatory infiltrate comprised of mononuclear cells and neutrophils. In a co-infection situation, although these cells would be unable to phagocyte and kill S. suis, they are highly activated by this pathogen. S. suis is not considered a primary pulmonary pathogen, but an exacerbated production of proinflammatory mediators during a co-infection with influenza virus may be important in the pathogenesis and clinical outcome of S. suis-induced respiratory diseases.

Transcriptome analysis and crop improvement (a review)

The identification and characterization of differential gene expression from tissues subjected to stress has gained much attention in plant research. The recognition of elements involved in the response to a particular stress enhances the possibility of promoting crop improvement through direct genetic modification. However, the performance of some of the 'first generation' of transgenic plants with the incorporation of a single gene has not always been as expected. These results have stimulated the development of new transgenic constructions introducing more than one gene and capable of modifying complex pathways. Several techniques are available to conduct the analysis of gene regulation, with such information providing the basis for novel constructs specifically designed to modify metabolism. This review deals with techniques that allow the identification and characterization of differentially-expressed genes and the use of molecular pathway information to produce transgenic plants.

Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays

Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a beta-fructofuranosidase, a fructokinase, an alpha-glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a beta-fructofuranosidase that could be used for scFOS degradation.

The transcriptome of Populus in elevated CO2

The consequences of increasing atmospheric carbon dioxide for long-term adaptation of forest ecosystems remain uncertain, with virtually no studies undertaken at the genetic level. A global analysis using cDNA microarrays was conducted following 6 yr exposure of Populus × euramericana (clone I-214) to elevated [CO2] in a FACE (free-air CO2 enrichment) experiment.• Gene expression was sensitive to elevated [CO2] but the response depended on the developmental age of the leaves, and < 50 transcripts differed significantly between different CO2 environments. For young leaves most differentially expressed genes were upregulated in elevated [CO2], while in semimature leaves most were downregulated in elevated [CO2].• For transcripts related only to the small subunit of Rubisco, upregulation in LPI 3 and downregulation in LPI 6 leaves in elevated CO2 was confirmed by anova. Similar patterns of gene expression for young leaves were also confirmed independently across year 3 and year 6 microarray data, and using real-time RT–PCR.• This study provides the first clues to the long-term genetic expression changes that may occur during long-term plant response to elevated CO2.

Root transcriptional responses of two melon genotypes with contrasting resistance to Monosporascus cannonballus (Pollack et Uecker) infection

Background: Monosporascus cannonballus is the main causal agent of melon vine decline disease. Several studies have been carried out mainly focused on the study of the penetration of this pathogen into melon roots, the evaluation of symptoms severity on infected roots, and screening assays for breeding programs. However, a detailed molecular view on the early interaction between M. cannonballus and melon roots in either susceptible or resistant genotypes is lacking. In the present study, we used a melon oligo-based microarray to investigate the gene expression responses of two melon genotypes, Cucumis melo 'Piel de sapo' ('PS') and C. melo 'Pat 81', with contrasting resistance to the disease. This study was carried out at 1 and 3 days after infection (DPI) by M. cannonballus. Results: Our results indicate a dissimilar behavior of the susceptible vs. the resistant genotypes from 1 to 3 DPI. 'PS' responded with a more rapid infection response than 'Pat 81' at 1 DPI. At 3 DPI the total number of differentially expressed genes identified in 'PS' declined from 451 to 359, while the total number of differentially expressed transcripts in 'Pat 81' increased from 187 to 849. Several deregulated transcripts coded for components of Ca2+ and jasmonic acid (JA) signalling pathways, as well as for other proteins related to defence mechanisms. Transcriptional differences in the activation of the JA-mediated response in 'Pat 81' compared to 'PS' suggested that JA response might be partially responsible for their observed differences in resistance. Conclusions: As a result of this study we have identified for the first time a set of candidate genes involved in the root response to the infection of the pathogen causing melon vine decline. This information is useful for understanding the disease progression and resistance mechanisms few days after inoculation.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Early Changes in Gene Expression Induced by Tobacco Smoke: Evidence for the Importance of Estrogen within Lung Tissue

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17 beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E2 within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer. Cancer Prev Res; 3(6); 707-17. (C) 2010 AACR.

Transcriptional Response to Hypoxia in the Aquatic Fungus Blastocladiella emersonii

Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly down-regulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1 alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes.

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)