Project description:Thicklip grey mullet hepatic transcriptome comparission from PASAIA harbour, Basque Country Active biomonitoring using sentinel species caged in differentially polluted aquatic environments is being implemented worldwide. To determine the biological effects of pollutants in a heavily polluted Basque harbour (Pasaia, 431700N 015500W), two cages specially designed to deploy thicklip grey mullets (Chelon labrosus) were placed in the inner- and outer-harbour. Cages were deployed for 5 and 21 days to determine possible differences between sites, linked to specific chemical burdens. Hepatic gene expression profiles were studied using Q-PCR and a toxicology tailored low density mullet microarray (160 genes). Chemical analysis showed high concentrations of metals, PAHs, PCBs, phthalates, and organometallic compounds at both sites, most chemicals showing slightly higher concentrations in the inner-harbour. Q-PCR expression studies confirmed chemical data as metallothionein and CYP1A showed significant expression upregulation at day 21 at both sites, but showed no differences between sites. The microarray showed gene expression profiles that were able to distinguish mullets according to the site they were deployed in and the day they were captured. Regarding sites, 39 genes showed significant differences at p<0.05 on the last sampling day. Two families of genes are to be highlighted. Phase I and II biotransformation genes CYP2, CYP3 and UGT were upregulated in the inner-harbour together with AhR2 and AhRR. Similarly, TBT binding protein and genes involved in lipid metabolism such us PPARa and CYP7 were upregulated. The latter two genes have also been reported to be regulated by TBT. In summary, the low density microarray designed for mullets proved to be useful to unveil even slight differences in pollutant burdens in field conditions. 24 samples:6 hepatic samples from Inner harbour after 3 days of exposure, 6 hepatic samples from outer harbour after 3 days of exposure, 6 hepatic samples from Inner harbour after 15 days of exposure and 6 hepatic samples from outer harbour after 15 days of exposure

Project description:The widely distributed thicklip grey mullet (Chelon labrosus) has been proposed as a suitable sentinel of pollution since it is able to survive heavily polluted marine/estuarine waters. Previous studies applying molecular to histological level biomarkers have indicated that mullets respond to exposure to chemical compounds. Microarrays are used to identify gene pathways responsive to specific chemical exposures. In this context, fragments of 129 genes relevant in peroxisome proliferation, detoxification, lipid metabolism, inflammatory/immune response, metal sequestration, oxidative and general stress, cell cycle regulation and proliferation, apoptosis, protein synthesis/degradation, and endocrine disruption were cloned through homological cloning using degenerate primers for microchip creation. Additional 31 sequences available in databases belonging to mugilid fishes were included in the final Agilent custom-microchip design. Female multitissue transcritome analysis was performed in mullets from Ondarrua. 108 genes showed differential expression when comparing female brain, gonad, gill and liver. Typical brain transcripts such as aromatase or dopamine receptor were expressed preferentially in the brain, whereas liver specific genes were detected in the liver; choriogenin-L, vitellogenins, fibrinogens or hepatocyte growth-factor. Genes related to peroxisome proliferation were systematically overrepresented in gonads. Female thicklip grey mullet tissue transcriptome comparission from Ondarrua, Basque Country GSM861357-GSM861379: 23 samples: 5 female samples from Gonad: female-Ondarru (F-L); 6 female samples from gonad: female-Ondarru (F-GO); 6 female samples from gill: female-Ondarru (F-GI); 6 female samples from brain: female-Ondarru (F-B); GSM886139-GSM886153: 15 samples: 2 intersex samples from Ondarru (I-L); 2 intersex gonad samples: intersex-Ondarru (I-GO); 6 female hepatic samples: female-Arriluze (F-A); 5 male hepatic samples: male-Arriluze (M-A) Immature Thicklip grey mullets were exposed to accetone, cadmium, benzo(a)pyrene and to nonylphenol and hepatic transcriptome was compared GSM886154-GSM886183: 30 samples: immature thicklip grey mullets hepatic samples 6 control seawater exposed samples, 6 acetone exposed samples; 6 cadmium exposed samples, 6 benzo(a)pyrene exposed samples (BAP) and 6 nonylphenol (NP) exposed samples.

Project description:The activated fibroblast is the central effector cell for the progressive fibrotic process that characterizes idiopathic pulmonary fibrosis (IPF). An understanding of the genomic phenotype of this cell in isolation is essential to the understanding of disease pathogenesis and is integral to strategizing therapeutic trials. Employing a unique technique that minimizes cellular phenotypic alterations, we characterized the genomic phenotype of non-cultured pulmonary fibroblasts from the lungs of patients with advanced IPF. This approach revealed several novel genes and pathways previously unreported in IPF fibroblasts. Specifically, we demonstrate altered expression in proteasomal constituents, ubiquitination mediators, the Wnt pathway and several cell cycle regulators suggestive of loss of normal cell cycle controls. The pro-inflammatory cytokine CXCL12 was also up-regulated which may provide a mechanism for fibrocytes’ recruitment, while up-regulated oncogenic KIT may promote fibroblast over proliferation. Paradoxically, pro-apoptotic inducers such as death inducing ligand TRAIL (TNFSF10) and pro-apoptotic Bax were also up-regulated. This comprehensive description of altered gene expression within IPF fibroblasts sheds further light on the complex interactions that characterize IPF. Further studies including therapeutic interventions directed at these pathways hold promise for the treatment of this devastating disease. 58 samples of total RNA isolated from 12 lungs of patients with end-stage idiopathic pulmonary fibrosis and 6 donors of normal lungs (controls) who were designated brain dead, non-diseased donors whose lungs failed criteria for transplantation and who were organ donors for research. RNA extraction followed the Qiagen RNeasy Kit using QIshredder columns for shredding of DNA contiminants. Experimental/control samples were amplified amino-allylated RNA labeled with Cy5 and Stratagene Reference RNA was amplified and amino-allylated and labeled with Cy3. Amplification was one round using Ambion MessageAmp II kit with amino-allylated UTP according to the protocol of the Duke University Institute for Genome Sciences and Policy. Amplification and amino-allylation of the Stratagene Reference RNA and Hybridization of Reference with patient samples and controls was done by the Duke Institute for Genomic Sciences and Policy.

Project description:Accidental spills of chemical or oil tankers such as Levoli Sun or Prestige have recently impacted the European seas. The analysis of the genes regulated by exposure to spilled chemical substances could help elucidating adaptatory and compensatory pathways that may participate in pathogenesis in marine biota. Hepatic expression profiles of male juvenile S. maximus exposed to a nÂº2 fuel-oil (FO) and to styrene were studied employing a turbot custom microarray containing 2715 3â-UTR biassed single-sequences, many of them unannotated. Hybridisations identified 169 and 293 genes significantly regulated after 3 and 14 days of exposure to FO, respectively. Typical PAH responsive genes such as AhRR, ARNT2, CYP1A1 and GST were significantly upregulated. Exposure significantly affected genes involved in protein metabolism, synthesis being the most regulated pathway with ribosomal proteins and various translation factors overexpressed. Protein synthesis could participate in compensatory pathways after FO induced protein damage. Additionally, two weeks of exposure resulted in the upregulation of ras pathway genes. Regarding styrene, gene expression was very variable within experimental groups and only 2 genes were significantly regulated after 3 days of exposure. After 1 week of exposure styrene modified the expression levels of 44 genes. Most highly regulated genes, that may constitute new biomarkers of exposure to styrene, included those coding for estrogen-responsive finger protein, transmembrane 9 superfamily member 4 or selenoprotein-T, but the only pathway found to be significantly regulated was once again that of protein synthesis. Funded EU project PRAGMA, Spanish MEC (CANCERMAR), Basque Government (Consolidated Research Groups GIC07/26-IT-393-07). Turbots were exposed to fuel-oil and to seawater control: 7 turbots in Control tank and 8 in Oiled tank. After 3 days of exposure turbots were in situ dissected and liver was extracted, inmersed in RNAlater and frozen in liquid N2

Project description:Accidental spills of chemical or oil tankers such as Levoli Sun or Prestige have recently impacted the European seas. The analysis of the genes regulated by exposure to spilled chemical substances could help elucidating adaptatory and compensatory pathways that may participate in pathogenesis in marine biota. Hepatic expression profiles of male juvenile S. maximus exposed to a nÂº2 fuel-oil (FO) and to styrene were studied employing a turbot custom microarray containing 2715 3â-UTR biassed single-sequences, many of them unannotated. Hybridisations identified 169 and 293 genes significantly regulated after 3 and 14 days of exposure to FO, respectively. Typical PAH responsive genes such as AhRR, ARNT2, CYP1A1 and GST were significantly upregulated. Exposure significantly affected genes involved in protein metabolism, synthesis being the most regulated pathway with ribosomal proteins and various translation factors overexpressed. Protein synthesis could participate in compensatory pathways after FO induced protein damage. Additionally, two weeks of exposure resulted in the upregulation of ras pathway genes. Regarding styrene, gene expression was very variable within experimental groups and only 2 genes were significantly regulated after 3 days of exposure. After 1 week of exposure styrene modified the expression levels of 44 genes. Most highly regulated genes, that may constitute new biomarkers of exposure to styrene, included those coding for estrogen-responsive finger protein, transmembrane 9 superfamily member 4 or selenoprotein-T, but the only pathway found to be significantly regulated was once again that of protein synthesis. Funded EU project PRAGMA, Spanish MEC (CANCERMAR), Basque Government (Consolidated Research Groups GIC07/26-IT-393-07). Turbots were exposed to fuel-oil and to seawater control: 7 turbots in Control tank and 8 in Oiled tank. After 14 days of exposure turbots were in situ dissected and liver was extracted, inmersed in RNAlater and frozen in liquid N2

Project description:Organotin compounds are highly persistent organic pollutants that bioaccumulate in marine biota, behaving as potent endocrine disruptors. Tributyltin (TBT) has been described as an environmental obesogen, as it contributes in mammals to lipid accumulation in a mechanism mediated by PPARÎ³ and RXR. With the aim of studying the molecular mechanisms that elicit TBT-induced pathogenesis in fish, thicklip grey mullets Chelon labrosus were exposed to low (10 ng/L) and high (500 ng/L) TBT concentrations for 1, 7 and 21 days, and gene transcription and metabolome profiles were studied. With this purpose, the multitissue transcriptome of mullet was sequenced by 454 pyrosequencing obtaining 126 Mb of sequence information. Assembly and annotation allowed spotting 8330 gene signatures on an oligonucleotide microarray that was used to identify hepatic gene transcription profiles specific to each TBT exposure set-up. Functional pathway analysis revealed that early profiles were characterized by genes related to response to organic substances and to oxidative stress and glucose metabolic processes. After 21 days of exposure, enriched GO terms in both concentrations were related to lipid homeostasis with a significant regulation of steroid metabolic and cholesterol biosynthetic processes. Also, the androgen receptor signalling pathway was regulated while PPAR signalling appeared as the most significantly regulated KEGG pathway. Neutral lipid accumulation measured by Oil-Red-O histochemistry did not show any treatment-related effect but hepatic and plasma NMR and GC-MS metabolomic analysis revealed distinctive metabolites. Aqueous profiles were characterised by lactate, glucose and alanine while GC-MS revealed a whole set of discriminating polyunsaturated fatty acids. Thus, TBT pathogenesis involves alterations of lipid metabolism through a mechanism that could involve PPARÎ³-regulated processes confirming the obesogen hypothesis for TBT in fish. Thicklip grey mullets were exposed to tributyltin (TBT). 3 treatments: control, TBT low dose (10ng/L), TBT high dose (500ng/L); 3 sampling times: 1 day, 1 week, 3 weeks. 6 mullets were dissected in each sampling point and group.

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Gene expression in adrenal medulla and kidney of a conditional mutant mouse in the SdhD gene

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