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  1. Abstract

    CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

     
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  2. Abstract

    The cellular stress response (CSR) is pervasive to all domains of life. It has shaped the interaction between organisms and their environment since the origin of the first cell. Although the CSR has been subject to a myriad of nuanced modifications in the various branches of life present today, its core features remain preserved. The scientific literature covering the CSR is enormous and the broad scope of this brief overview was challenging. However, it is critical to conceptually understand how cells respond to stress in a holistic sense and to point out how fundamental aspects of the CSR framework are integrated. It was necessary to be extremely selective and not feasible to even mention many interesting and important developments in this expansive field. The purpose of this overview is to sketch out general and emerging CSR concepts with an emphasis on the initial cellular strain resulting from stress (macromolecular damage) and the evolutionarily most highly conserved elements of the CSR. Examples emphasize fish and aquatic invertebrates to highlight what is known in organisms beyond mammals, yeast, and other common models. Nonetheless, select pioneering studies using canonical models are also considered and the concepts discussed are applicable to all cells. More detail on important aspects of the CSR in aquatic animals is provided in the accompanying articles of this special issue.

     
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  3. Abstract

    Stress represents a multi‐faceted force that is central for the evolution of life. Organisms evolve while adapting to stress and stressful contexts often represent selective bottlenecks. To understand stress effects on biological systems and corresponding coping strategies it is imperative to properly define stress and the resulting strain that triggers compensatory responses in cells and organisms. Here I am deriving such definitions for biological systems based on principles that are established in physics. The relationship between homeostasis of critical biological variables, the elastic limit, the cellular stress response (CSR), cellular homeostasis response (CHR), system dysregulation, and the breaking point (death of the system) is outlined. Dysregulation of homeostatic set‐points of biological variables perturbs the functional properties of the system, shifting them out of the evolutionarily optimized range. Such shifts are accompanied by elevated rates of macromolecular damage, which represents a nonspecific signal for induction of a universal response, the CSR. The CSR complements the CHR in re‐establishing homeostasis of the system as a whole. Moreover, the CSR is essential for coping with suboptimal conditions while the system is in a dysregulated state and for removing excessive damage that accumulates during such periods. The extreme complexity of biological systems and their emergent properties often necessitate monitoring stress effects on many biological variables simultaneously to properly deduce stress effects on the system as a whole. Therefore, increased utilization of systems biology (omics) approaches for characterizing cellular and organismal stress responses facilitates the reductionist dissection of biological stress response mechanisms.

     
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  4. Abstract

    Salinity stress occurs when salt concentration in the environment changes rapidly, for example because of tidal water flow, rainstorms, drought, or evaporation from small bodies of water. However, gradual changes in salt concentration can also cause osmotic stress in aquatic habitats if levels breach thresholds that reduce the fitness of resident organisms. The latter scenario is exemplified by climate change driven salinization of estuaries and by dilution of ocean surface salinity through changes in the water cycle. In this review, we discuss how fish employ the evolutionarily conserved cellular stress response (CSR) to cope with these different forms of salinity stress. Macromolecular damage is identified as the cause of impaired physiological performance during salinity stress and serves as the signal for inducing a CSR. Basic aspects of the CSR have been observed in fish exposed to salinity stress, including repair and protection of cellular macromolecules, reallocation of energy, cell cycle arrest, and in severe cases, programmed cell death. Osmosensing and signal transduction events that regulate these aspects of the CSR provide a link between environmental salinity and adaptive physiological change required for survival. The CSR has evolved to broaden the range of salinities tolerated by certain euryhaline fish species, but is constrained in stenohaline species that are sensitive to changes in environmental salinity. Knowledge of how the CSR diverges between euryhaline and stenohaline fish enables understanding of physiological mechanisms that underlie salt tolerance and facilitates predictions as to the relative vulnerabilities of different fish species to a rapidly changing hydrosphere.

     
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  5. 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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  6. 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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  7. null (Ed.)
    Cell cultures are effective supplemental models to study specific biochemical pathways used for environmental adaption in animals. They enable isolation from system influence and facilitate control the extracellular environment. For work focusing on fish species many representative cell lines now exist, including a tilapia brain cell line (OmB) developed in our lab. CRISPR/Cas9 gene editing is an additional tool aiding these studies by allowing manipulation of specific genetic loci and evaluating their causal relationship between phenotypes of interest. However, established CRISPR/Cas9 gene targeting tools and methods often have not functioned as efficiently in fish cells as seen in other animal cell models such as mammalian cell lines, consistent with our initial attempts to apply CRISPR/Cas9 in OmB cells that failed to indicate genomic alteration at the targeted sites. Poor expression of heterologous promoters in OmB cells was hypothesized to be a primary cause for this occurrence so we constructed a custom plasmid vector based system utilizing tilapia endogenous promoters (EF1 alpha to express Cas9 and a U6 to express gRNAs). This system demonstrated substantial editing of most target sites attempted with mutational efficiency as high 80%. This work specifically highlighted the importance of phylogenetic proximity in selection of a polymerase III promoter for gRNA expression as commonly used interspecies U6 promoters (human and zebrafish) yielded no detectable gene editing when applied in this system with a common gRNA target sequence. These new tools will allow generation of knockout cell lines for gene targeting studies in tilapia and other phylogenetically close fish species. 
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  8. 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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  9. null (Ed.)
    Histone post-translational modifications (PTMs) are epigenetic marks that modify the state of chromatin and lead to alterations in gene expression. Advances in mass spectrometry have enabled the high-throughput analysis of histone PTMs without the need for prior knowledge of individual PTMs of interest. In this study, the global histone PTM landscape was analyzed in the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus) through tandem mass spectrometry using data dependent acquisition (DDA-LCMS2) and PTM mapping approaches. PTM assignment to a specific amino acid was validated using A-score and localization probability scores that are based on the detection of diagnostic MSMS ions. These values signify the robustness of PTM assignment to a specific residue within the protein sequence. For PTMs that were represented by both modified and unmodified versions of the corresponding peptide, the stoichiometry was calculated and compared between tissues. We have identified multiple types of histone PTMs and assigned them to specific residues in each tissue. These PTMs include acetylation, methylation, demethylation, trimethylation, phosphorylation/ dehydration, and ubiquitination. Our results indicate that the gills, kidney, and testes each display a unique profile of histone PTMs. These data provide a strong basis for the generation of spectral libraries that enable high-throughput quantitative analyses of histone PTM stoichiometry on a global scale in tilapia exposed to diverse environmental and developmental contexts. 
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