Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Oligonucleotide microarray for Sparus aurata

ABSTRACT: This study describes the development and validation of the Aquagenomic Sparus aurata oligonucleotide-microarray (SAQ) based on the Agilent Technology system (eArray) to provide a platform for studies on the gene expression of gilthead seabream. The platform developed used all available public ESTs stored and annotated in the Aquagenomic Consortium seabream library (10K). In fish, lipopolysaccharide (LPS) gives a robust cytokine response that is stimulated by crude LPS preparations, some component of the LPS complex are responsible for this stimulation. Peptidoglycan (PGN) is a component of G-negative bacteria (found as a contaminant of crude LPS preparations) able to be recognized by macrophages inducing depth transcriptional modulations and a strong inflammatory response. For microarray analysis, head kidney macrophage cultures were used (N = 36 fish). Each cell culture was stimulated with equal concentrations of PGN and LPS from E. coli O111:B4 strain (10 ug/mL): non-stimulated cell cultures (control n = 9 fish), stimulated during 6 h with LPS (n = 9), and stimulated during 1 h (n = 9), and 6 h (n = 9) with PGN. A loop microarray design approach was used for the study. All experimental RNA samples were labelled with a single colour dye (Cy3) and each stimulated sample was compared to the control sample (pool without stimulation) labelled with the same dye (Cy3). Our microarray analyses identified differential transcriptional modulations in macrophages stimulated with both LPS and PGN at the level of differentially activated RNA transcripts related with the regulation of transcriptional program, prostaglandin synthesis or highlighting the expression of responsive gene-cassettes tightly related to LPS-PGN host recognition. A total of 43,398 oligonucleotide probes were used to construct a high-density seabream microarray based on the Agilent 4 × 44 K design format. Microarray hybridization validation was made by analyzing the gene expression profiles in primary cultures of seabream macrophages (MC). 7,285 transcripts with annotated sequences were spotted in triplicate onto the slide (total probes 21,855), as well as 8,377 ESTs without annotation, 183 enriched sequences (gene bank) with 15 replicated probes (total probes 2,745), and finally 1,417 internal control probes of Agilent (N = 43,398).

ORGANISM(S): Sparus aurata

SUBMITTER: Sebastian Boltana

PROVIDER: E-GEOD-28610 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Fish respond to stressors by means of an array of changes that involve many biological compartments including molecular, cellular, metabolic, physiological and behavioral acclimation. Among these changes, the immune system also experiences significant alterations that are characterized by an induction of the innate non-specific responses and changes in the acquired specific response. The present investigation was undertaken to check whether the innate response of the complement system is affected after a combination of an immune challenge and a husbandry stressor. In European seabass, two different transcripts of the complement C3 protein, which correspond to two tissue-specific isoforms, have been detected in liver. They may come from post-transcriptional modifications at the pre-mRNA level or from alternative splicing in order to generate a diversified repertoire of protein isoforms in some fish species according to the tissue were they are located. After cloning the hepatic C3, the quantification of seabass isoforms showed specific responses, thus confirming the possible specialized roles of the C3 different isoforms in terms of defence immunity. On one hand, a stronger esbC3_2 expression was observed in front to the viral challenge, suggesting a specific participation of this molecule in front to viruses. This observation is in agreement with previous studies in trout macrophages in our lab, demonstrating that the immune response to viral infections was stronger than to bacterial ones. On the other hand, the esbC3_1 expression appeared stronger after bacterial challenge, especially under high density conditions. Our results confirm the hypothesis of an immune-activation of specific isoforms of the C3 protein of the complement system as a non-specific response in front to either immune or other type of stressors. In order to confirm our hypothesis, we created an oligonucleotide microarray (Agilent Technologies) containing 6275 annotated transcripts, each with x3 specific probes, and 4516 ESTs with 1 probe/target sequence. Gene expression profiles obtained from the livers of fish held under high density culture conditions highlighted a significant contrast in the liver response. In total, 54 transcripts were differentially expressed (GDE; fold change FC >2) in treatment over the control (p<0.01)/201 (p<0.05). Oligonucleotide probes were used to construct a high-density seabass microarray based on the Agilent 4 × 44 K design format (http://www.agilent.com/), which covers 13,199 unique transcripts of Dicentrarchus labrax. Oligonucleotides, 60-mer plus a “linker”, are synthesized directly on glass slides using the Agilent’s SurePrint Tecnology; oligo design was carried out by Agilent and three or more non-overlapping probes were designed for each EST cluster. The number of transcripts represented by three is 6,275, while 6,924 targets EST have only one probe.

Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.

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Oligonucleotide microarray for Sparus aurata

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