ae57a643dc3716c0ab8453d4b75adb0c pmc_28165358.pdf 0ebb52e9f99fd1ff99e0a85868420c5be11e5a19 pmc_28165358.pdf 316e67c0e20dc6ce4987442f2c9857191c592af093078cff495eaaf5a63bcfa7 pmc_28165358.pdf Title: Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs Subject: This study describes the development and validation of an enriched oligonucleotide- microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response. Keywords: expressed sequence tags (EST); oligo-nucleotide microarray; pathogen-associated molecular patterns (PAMPs); lipopolysaccharide (LPS); peptidoglycan (PGN); macrophages Author: Sebastian Boltaña, Barbara Castellana, Giles Goetz, Lluis Tort, Mariana Teles, Victor Mulero, Beatriz Novoa, Antonio Figueras, Frederick W. Goetz, Cristian Gallardo-Escarate, Josep V. 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CEST Size: 4954555 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: ぉのぴひはつふっぴどはの〠 Item: Title: げづびふぬぴび〠 Children: Item: Title: ぉつづのぴどてどっちぴどはの〠はて〠ぉねねふのづ〭げづぬちぴづつ〠ぅへばひづびびづつ〠こづぱふづのっづ〠ごちでび〠〨ぅこご〩〠 Item: Title: けふちぬどぴべ〠ぁびびづびびねづのぴ〠はて〠ぴとづ〠きどっひはちひひちべ〠えべぢひどつどびちぴどはの〠 Item: Title: ぃはねばちひちぴどぶづ〠ごひちのびっひどばぴはねづ〠ぁのちぬべびどび〠 Item: Title: いどびっふびびどはの〠 Children: Item: Title: ぉつづのぴどてどっちぴどはの〠はて〠ぉねねふのづ〭げづぬちぴづつ〠ごひちのびっひどばぴび〠どの〠こ〮〠ちふひちぴち〠 Item: Title: いづぶづぬはばねづのぴ〠はて〠こ〮〠ちふひちぴち〠くぎき〠 Item: Title: 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