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  1. null (Ed.)
    Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 in tilapia gills was reproduced in the tilapia OmB cell neuroepithelial cell line. OSTF1 shares the signature sequence of the TSC-22 family suggesting that it is a transcriptional repressor. If, in fact, OSTF1 is a transcription factor, we hypothesize that it will localize to the nucleus during hyperosmotic stress. Using standard cloning procedures, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C- or N-terminus. Using fluorescent microscopy we show that the fusion proteins are retained in the cytosol under iso-osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope with automated scanning stage. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates influence of specific OSTF1 domains on nuclear localization. 
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  2. null (Ed.)
    Interactions of organisms with their environment are complex and regulation at different levels of biological organization from genotype to phenotype is often non-linear. While studies of transcriptome regulation are now common for many species, corresponding quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed, demonstrating the complementary nature of the DIA assay library approach and suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. In addition to significance testing, the application of functional enrichment analyses using STRING and KEGG to DIA assay datasets identified myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. In conclusion, this study presents an innovative approach for targeted quantitative proteomics used to identify proteins and biological processes that are regulated non-linearly at mRNA and protein levels during a change of environmental salinity. 
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  3. null (Ed.)
    Interactions of organisms with their environment are complex and regulation at different levels of biological organization from genotype to phenotype is often non-linear. While studies of transcriptome regulation are now common for many species, corresponding quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed, demonstrating the complementary nature of the DIA assay library approach and suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. In addition to significance testing, the application of functional enrichment analyses using STRING and KEGG to DIA assay datasets identified myo-inositol metabolism, antioxidant and xenobiotic functions, and signaling mechanisms as key elements controlled by salinity in tilapia kidneys. In conclusion, this study presents an innovative approach for targeted quantitative proteomics used to identify proteins and biological processes that are regulated non-linearly at mRNA and protein levels during a change of environmental salinity. 
    more » « less