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Int. J. Mol. Sci. 2017, 18(2), 317; doi:10.3390/ijms18020317

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

1
Institute of Biotechnology and Biomedicine, University Autónoma de Barcelona, Barcelona 08193, Spain
2
University of Stirling, School of Natural Sciences, Stirling FK9 4LA, Scotland, UK
3
Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR), Biotechnology Center, University of Concepción, Concepción 4030000, Chile
4
The Child & Family Research Institute, Vancouver, BC V5Z 4H4, Canada
5
Department of Obstetrics & Gynecology, The University of British Columbia, Vancouver, BC V6T 1Z4, Canada
6
Northwest Fisheries Science Centre, Seattle, WA 98112, USA
7
Department Biologia Cellular, Immunologia I Fisiologia, Universitat Autónoma de Barcelona, Barcelona 08193, Spain
8
Department de Biología Celular e Histología, Facultad de Biología, Universidad de Murcia, Murcia 30100, Spain
9
Institute of Marine Research, Spanish National Research Council (CSIC), Eduardo Cabello 6, Vigo 36208, Spain
10
Department of Physiology and Immunology, University of Barcelona, Barcelona 08028, Spain
*
Author to whom correspondence should be addressed.
Academic Editors: Céline Audet and Li Lin
Received: 17 May 2016 / Revised: 31 August 2016 / Accepted: 8 October 2016 / Published: 3 February 2017
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
View Full-Text   |   Download PDF [4838 KB, uploaded 3 February 2017]   |  

Abstract

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. View Full-Text
Keywords: expressed sequence tags (EST); oligo-nucleotide microarray; pathogen-associated molecular patterns (PAMPs); lipopolysaccharide (LPS); peptidoglycan (PGN); macrophages expressed sequence tags (EST); oligo-nucleotide microarray; pathogen-associated molecular patterns (PAMPs); lipopolysaccharide (LPS); peptidoglycan (PGN); macrophages
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Boltaña, S.; Castellana, B.; Goetz, G.; Tort, L.; Teles, M.; Mulero, V.; Novoa, B.; Figueras, A.; Goetz, F.W.; Gallardo-Escarate, C.; Planas, J.V.; Mackenzie, S. 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. Int. J. Mol. Sci. 2017, 18, 317.

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