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Introduction: Bone marrow embolization may complicate orthopedic surgery, potentially causing fat embolism syndrome. The inflammatory potential of bone marrow emboli is unclear. We aimed to investigate the inflammatory response to femoral intramedullary nailing, specifically the systemic inflammatory effects in plasma, and local tissue responses. Additionally, the plasma response was compared to that following intravenous injection of autologous bone marrow. Methods: Twelve pigs underwent femoral nailing (previously shown to have fat emboli in lung and heart), four received intravenous bone marrow, and four served as sham controls. Blood samples were collected hourly and tissue samples postmortem. Additionally, we incubated bone marrow and blood, separately and in combination, from six pigs in vitro. Complement activation was detected by C3a and the terminal C5b-9 complement complex (TCC), and the cytokines TNF, IL-1ß, IL-6 and IL-10 as well as the thrombin-antithrombin complexes (TAT) were all measured using enzyme-immunoassays. Results: After nailing, plasma IL-6 rose 21-fold, compared to a 4-fold rise in sham (p=0.0004). No plasma differences in the rest of the inflammatory markers were noted across groups. However, nailing yielded 2-3-times higher C3a, TCC, TNF, IL-1ß and IL-10 in lung tissue compared to sham (p<0.0001-0.03). Similarly, heart tissue exhibited 2-times higher TCC and IL-1ß compared to sham (p<0.0001-0.03). Intravenous bone marrow yielded 8-times higher TAT than sham at 30 minutes (p<0.0001). In vitro, incubation of bone marrow for four hours resulted in 95-times higher IL-6 compared to whole blood (p=0.03). Discussion: A selective increase in plasma IL-6 was observed following femoral nailing, whereas lung and heart tissues revealed a broad local inflammatory response not reflected systemically. In vitro experiments may imply bone marrow to be the primary IL-6 source.
Assuntos
Embolia Gordurosa , Interleucina-6 , Pulmão , Animais , Suínos , Interleucina-6/sangue , Embolia Gordurosa/etiologia , Embolia Gordurosa/sangue , Embolia Gordurosa/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/metabolismo , Medula Óssea/metabolismo , Fixação Intramedular de Fraturas/efeitos adversos , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/imunologia , Inflamação/sangue , Inflamação/imunologia , Feminino , Citocinas/sangue , Citocinas/metabolismo , Pinos Ortopédicos , Ativação do Complemento , Fêmur/metabolismo , Modelos Animais de DoençasRESUMO
Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.
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Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.
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Plaquetas , Complemento C3b , Escherichia coli , Agregação Plaquetária , Humanos , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Complemento C3b/imunologia , Hirudinas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Técnicas In VitroRESUMO
BACKGROUND: The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. RESULTS: We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. CONCLUSION: ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron-exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.
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Antozoários/genética , Genoma Mitocondrial/genética , Íntrons/genética , Mitocôndrias/genética , Splicing de RNA/genética , RNA Mitocondrial/genética , Animais , Precursores de RNARESUMO
Ocean acidification threatens to disrupt interactions between organisms throughout marine ecosystems. The diversity of reef-building organisms decreases as seawater CO2 increases along natural gradients, yet soft-bodied animals, such as sea anemones, are often resilient. We sequenced the polyA-enriched transcriptome of adult sea anemone Anemonia viridis and its dinoflagellate symbiont sampled along a natural CO2 gradient in Italy to assess stress levels in these organisms. We found that about 3.1% of the anemone transcripts, but <1% of the Symbiodinium sp. transcripts were differentially expressed. Processes enriched at high seawater CO2 were linked to cellular stress and inflammation, including significant up-regulation of protective cellular functions and down-regulation of metabolic pathways. Transposable elements were differentially expressed at high seawater CO2, with an extreme up-regulation (> 100-fold) of the BEL-family of long terminal repeat retrotransposons. Seawater acidified by CO2 generated a significant stress reaction in A. viridis, but no bleaching was observed and Symbiodinium sp. appeared to be less affected. These observed changes indicate the mechanisms by which A. viridis acclimate to survive chronic exposure to ocean acidification conditions. We conclude that many organisms that are common in acidified conditions may nevertheless incur costs due to hypercapnia and/or lowered carbonate saturation states.
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Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21â¯kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates. Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying the conserved cytochrome oxidase II gene and a second allele of the small subunit ribosomal RNA gene.
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Cromossomos , Genoma Mitocondrial , Anêmonas-do-Mar/genética , Animais , Evolução Biológica , Complexo IV da Cadeia de Transporte de Elétrons/genética , Transferência Genética Horizontal , Íntrons , Fosforilação Oxidativa , Filogenia , RNA Ribossômico/genéticaRESUMO
A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.
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DNA Mitocondrial/genética , Gadus morhua/genética , RNA Longo não Codificante/genética , Sequências de Repetição em Tandem , Animais , Herança Materna , Polimorfismo GenéticoRESUMO
OBJECTIVE: The objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes. RESULTS: We determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species' distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.
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DNA Mitocondrial/genética , Gadus morhua/genética , Animais , Genoma , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
A notable feature of hexacoral mitogenomes is the presence of complex self-catalytic group I introns. We investigated mitogenome structural variations and evolutionary mechanisms in actiniarian sea anemones based on the complete mitogenome sequence of the cold-water sea anemone species Urticina eques, Bolocera tuediae, Hormathia digitata and Metridium senile, and two isolates of the sub-tropical Aiptasia pulchella. Whole genome sequencing at 50 times coverage of B. tuediae and H. digitata indicated low mtDNA copy number of per haploid nuclear genome and presence of rare haplotypes. A group I intron inserted in ND5 was found to host essential mitochondrial protein genes in all species, and an additional truncated copy of ND5 in B. tuediae. A second group I intron (inserted in COI) that contained a homing endonuclease gene (HEG) was present in all mtDNA examined. Different variants of HEGs were observed, and included expressed elements fused in-frame with upstream exons and free-standing HEGs embedded within the intron. A notable hallmark of HEGs was a high extent of overlap with ribozyme structural elements; the U. eques HEG overlapped with the entire intron. We reconstructed the evolutionary history of the COI intron from insertion at unoccupied cognate sites, through HEG degradation, to intron loss. We also identified a novel insertion element in U. eques that contained two expressed protein-coding genes. An evolutionary analysis of the sea anemone mtDNA genes revealed higher substitution rates in the HEG and the insertion sequence as compared to the other loci, indicating relaxed selective pressures in these elements. We conclude that sea anemone mitogenomes are surprisingly dynamic in structure despite the economical organization and low sequence mutation rate.
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Elementos de DNA Transponíveis , Genoma Mitocondrial , Íntrons , Filogenia , Anêmonas-do-Mar/genética , Animais , Sequência de Bases , Evolução Biológica , DNA Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Mutagênese Insercional , Conformação de Ácido Nucleico , Análise de Sequência de DNARESUMO
Sequencing of mitochondrial gene fragments from specimens representing a wide range of geographical locations has indicated limited population structuring in Atlantic cod (Gadus morhua). We recently performed whole genome analysis based on next-generation sequencing of two pooled ecotype samples representing offshore migratory and inshore stationary cod from the North-east Atlantic Ocean. Here we report molecular features and variability of the 16.7kb mitogenome component that was collected from the datasets. These sequences represented more than 25 times coverage of each individual and more than 1100 times coverage of each ecotype sample. We estimated the mitogenome to have evolved 14 times more rapidly than the nuclear genome. Among the 365 single nucleotide polymorphism (SNP) sites identified, 121 were shared between ecotypes, and 151 and 93 were private within the migratory and stationary cod, respectively. We found 323 SNPs to be located in protein coding genes, of which 29 were non-synonymous. One synonymous site in ND2 was likely to be under positive selection. FST measurements indicated weak differentiation in ND1 and ND2 between ecotypes. We conclude that the Atlantic cod mitogenome and the nuclear genome apparently evolved by distinct evolutionary constraints, and that the reproductive isolation observed from whole genome analysis was not visible in the mtDNA sequences.
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Ecossistema , Evolução Molecular , Gadus morhua/genética , Variação Genética/genética , Genética Populacional , Genoma Mitocondrial/genética , Animais , Oceano Atlântico , Sequência de Bases , Dados de Sequência Molecular , NADH Desidrogenase/genética , Polimorfismo de Nucleotídeo Único/genética , Isolamento Reprodutivo , Seleção Genética , Análise de Sequência de DNARESUMO
Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co-occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome-wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next-generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single-nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North-east Atlantic.
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Migração Animal , Ecótipo , Gadus morhua/genética , Genética Populacional , Animais , Oceano Atlântico , Ligação Genética , Genótipo , Polimorfismo de Nucleotídeo Único , Isolamento Reprodutivo , Análise de Sequência de DNARESUMO
Marine bioprospecting is the search for new marine bioactive compounds and large-scale screening in extracts represents the traditional approach. Here, we report an alternative complementary protocol, called digital marine bioprospecting, based on deep sequencing of transcriptomes. We sequenced the transcriptomes from the adult polyp stage of two cold-water sea anemones, Bolocera tuediae and Hormathia digitata. We generated approximately 1.1 million quality-filtered sequencing reads by 454 pyrosequencing, which were assembled into approximately 120,000 contigs and 220,000 single reads. Based on annotation and gene ontology analysis we profiled the expressed mRNA transcripts according to known biological processes. As a proof-of-concept we identified polypeptide toxins with a potential blocking activity on sodium and potassium voltage-gated channels from digital transcriptome libraries.
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Regulação da Expressão Gênica/fisiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Anêmonas-do-Mar/metabolismo , Transcriptoma , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Animais , Temperatura Baixa , Biologia Computacional/métodos , Descoberta de Drogas , Ecossistema , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Dados de Sequência Molecular , Oceanos e Mares , Conformação Proteica , Anêmonas-do-Mar/químicaRESUMO
We present an initial genomic analysis of the non-symbiotic scleractinian coral Lophelia pertusa, the dominant cold-water reef-building coral species in the North Atlantic Ocean. A significant fraction of the deep sequencing reads was of mitochondrial and microbial origins. SOLiD deep sequencing reads from fragment library experiments of total DNA and PCR amplified mitogenome generated about 21,000 times and 136,000 times coverage, respectively, of the 16,150 bp mitogenome. Five polymorphic sites that include two non-synonymous sites in the NADH dehydrogenase subunit 5 genes were detected in both experiments. This observation is surprising since anthozoans in general exhibit very low mtDNA sequence variation at intraspecific level compared to nuclear sequences. More than fifty bacterial species associated with the coral isolate were also sequence detected, representing at least ten complete genomes. Most reads, however, were predicted to originate from the Lophelia nuclear genome.
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Antozoários/genética , Genoma , Polimorfismo Genético , Animais , Sequência de Bases , Mapeamento Cromossômico/métodos , DNA/genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Análise de Sequência de DNARESUMO
Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3' end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.
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Cnidários/genética , Genoma Mitocondrial , Íntrons , Filogenia , Animais , Sequência de Bases , Cnidários/classificação , DNA Mitocondrial/genética , Evolução Molecular , Ordem dos Genes , Padrões de Herança , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Análise de Sequência de DNARESUMO
The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.
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Gadus morhua/genética , Genoma/genética , Análise de Sequência de DNA/métodos , Animais , Biologia Computacional , Perfilação da Expressão Gênica , MicroRNAs/genéticaRESUMO
RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-generation sequencing platforms have recently been developed for de novo whole transcriptome analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics (sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential identification of all expressed genes in a sample. When combined to high-throughput bioinformatics and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral transcriptomes with the focus on cold-water sea anemones and related organisms.
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Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ensaios de Triagem em Larga Escala , RNA/genética , Anêmonas-do-Mar/genética , Análise de Sequência de RNA , Adaptação Fisiológica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Temperatura Baixa , Mitocôndrias/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oceanos e Mares , RNA/química , RNA/metabolismo , Anêmonas-do-Mar/citologia , Alinhamento de SequênciaRESUMO
The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.