Kelly H. Soanes

Sequence and expression of C-type lectin receptors in Atlantic salmon ( Salmo salar )

The diverse receptors of the C-type lectin superfamily play key roles in innate immunity. In mammals, cell surface receptors with C-type lectin domains are involved in pathogen recognition and in immune response, and in some cases are exploited by pathogens to gain entry into cells. This study reports on sequence and expression analysis of three paralogous group II C-type lectins from the teleost fish Atlantic salmon (Salmo salar). Each of the receptors showed similarity to immune-relevant mammalian receptors in terms of amino acid sequence and overall organization within the C-type lectin-like domain (CTLD). Two of the three have cytoplasmic motifs consistent with the immunoreceptor tyrosine-based activation motifs (ITAM), which are known to modulate downstream functions in leukocytes. All three C-type lectin receptors were expressed in multiple tissues of healthy fish, including peripheral blood leukocytes and salmon head kidney cells (SHK-1). Each receptor was up-regulated in salmon liver in response to infection by Aeromonas salmonicida and one receptor was substantially up-regulated in cultured SHK-1 cells in response to lipopolysaccharide (LPS). Putative binding sites for the CAAT-enhancer-binding protein (C/EBP) family of transcription factors in the regulatory regions of these C-type lectin genes may mediate their response to bacteria and LPS in salmon leukocytes. The identification of these types of receptors in distinct populations of cells within the immune system will provide important markers for identifying and categorizing the state of differentiation or activation of these cells and lead to further understanding of the interaction between the salmon host and multiple pathogens.

Distinct models of induced hyperactivity in zebrafish larvae

The analysis of behavioural hyperactivity can provide insights into how perturbations in normal activity may be linked to the altered function of the nervous system and possibly the symptoms of disease. As a small vertebrate zebrafish have numerous experimental advantages that are making them a powerful model for these types of studies. While the majority of behavioural studies have focused on adult zebrafish, it has become apparent that larvae can also display complex stereotypical patterns of behaviour. Here we have used three compounds (pentylenetetrazole (PTZ), aconitine and 4-aminopyridine) that have different neuronal targets (GABA, sodium and potassium channels), to induce distinct patterns of hyperactivity in larvae. Our studies have revealed that each compound produces a number of distinct concentration-dependent activity patterns. This work has shown for the first time that at sub-convulsive concentrations, PTZ can reverse the normal behavioural response to alternating periods of light and dark in zebrafish larvae. It also appears that both PTZ and 4-aminopyridine produce distinct changes in the normal startle response patterns immediately following light/dark transitions that may be the result of an elevation in stress/anxiety. Aconitine produces a general elevation in activity that eliminates the normal response to light and dark. In addition to differences in the patterns of behaviour each compound also produces a unique pattern of c-fos (an immediate early gene) expression in the brain. While more work is required to make direct links between region specific neuronal activity and individual behaviours, these models provide a framework with which to study and compare mechanistically different types of inducible behaviours.

A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics

Modelling neurological diseases has proven extraordinarily difficult due to the phenotypic complexity of each disorder. The zebrafish has become a useful model system with which to study abnormal neurological and behavioural activity and holds promise as a model of human disease. While most of the disease modelling using zebrafish has made use of adults, larvae hold tremendous promise for the high-throughput screening of potential therapeutics. The further development of larval disease models will strengthen their ability to contribute to the drug screening process. Here we have used zebrafish larvae to model the symptoms of bipolar disorder by treating larvae with sub-convulsive concentrations of the GABA antagonist pentylenetetrazol (PTZ). A number of therapeutics that act on different targets, in addition to those that have been used to treat bipolar disorder, were tested against this model to assess its predictive value. Carbamazepine, valproic acid, baclofen and honokiol, were found to oppose various aspects of the PTZ-induced changes in activity. Lidocaine and haloperidol exacerbated the PTZ-induced activity changes and sulpiride had no effect. By comparing the degree of phenotypic rescue with the mechanism of action of each therapeutic we have shown that the low-concentration PTZ model can produce a number of intermediate phenotypes that model symptoms of bipolar disorder, may be useful in modelling other disease states, and will help predict the efficacy of novel therapeutics.

Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.

The zebrafish embryo as a tool for screening and characterizing pleurocidin host-defense peptides as anti-cancer agents

SUMMARY The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.

Recombinant production and characterization of the carbohydrate recognition domain from Atlantic salmon C-type lectin receptor C (SCLRC).

The Atlantic salmon C-type lectin receptor C (SCLRC) locus encodes a potential oligomeric type II receptor. C-type lectins recognize carbohydrates in a Ca(2+)-dependent manner through structurally conserved, yet functionally diverse, C-type lectin-like domains (CTLDs). Many conserved amino acids in animal CTLDs are present in SCLRC, with the notable exception of an asparagine crucially involved in Ca(2+)- and carbohydrate-binding, which is tyrosine in SCLRC. SCLRC also contains six cysteines that form three disulfide bonds. Although SCLRC was originally identified as an up-regulated transcript responding to Aeromonas salmonicida infection, the biological role of this protein is still unknown. To study the structure and ligand binding properties of SCLRC, we created a homology model of the 17kDa CTLD and produced it as an affinity-tagged protein in the periplasm of Escherichia coli by co-expression of proteins that facilitate disulfide bond formation. The recombinant form of SCLRC was characterized by a protease protection assay, a solid-phase carbohydrate-binding assay, and frontal affinity chromatography. On the basis of this characterization, we classify SCLRC as a C-type lectin that binds to mannose and its derivatives.

Prohormone Processing in Zebrafish

Proprotein convertases (PCs) are secretory proteolytic enzymes that activate precursor proteins into biologically active forms by limited proteolysis at one or multiple internal sites. PCs are implicated in the processing of multiple protein precursors, including hormones, proteases, growth factors, angiogenic factors, and receptors. PCs have been linked recently to various pathologies such as Alzheimers disease, tumorigenesis, and infections. The zebrafish has emerged as an attractive model for studying the role of PCs not only in substrate production but also in development. Herein we describe methods that are used to characterize DNA sequences of PCs in zebrafish, as well as to evaluate the ontogeny and tissue distribution of their transcripts. We also provide information on the morpholino-mediated knockdown of proprotein convertases.

The eukaryotic Initiation Factor 6 (eIF6) regulates ecdysone biosynthesis by modulating translation in Drosophila

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.

Increasing Eukaryotic Initiation Factor 6 (eIF6) Gene Dosage Stimulates Global Translation and Induces a Transcriptional and Metabolic Rewiring that Blocks Programmed Cell Death

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.

The Drosophila Eukaryotic Initiation Factor eIF6 affects development by regulating apoptosis via the ecdysone pathway

During development, ribosome biogenesis and translation reach peak activities, due to impetuous cell proliferation. Current models predict that protein synthesis elevation is controlled by transcription factors and signalling pathways. Developmental models addressing translation factors overexpression effects are lacking. Eukaryotic Initiation Factor (eIF6) is necessary for ribosome biogenesis and efficient translation. eIF6 is a single gene, conserved from yeasts to mammals, suggesting a tight regulation need. We generated a Drosophila melanogaster in vivo model of eIF6 upregulation, demonstrating a boost in general translation and the shut off of the ecdysone biosynthetic pathway. Translation modulation in S2 cells showed that translational rate and ecdysone biosynthesis are inversely correlated. In vivo, eIF6-driven alterations delayed programmed cell death (PCD), resulting in aberrant phenotypes, partially rescued by ecdysone administration. Our data show that eIF6 triggers a translation program with far-reaching effects on metabolism and development, stressing the driving and central role of translation.Translation factors downregulation modulates gene expression but the effect of their overexpression is still unknown. The Eukaryotic Initiation Factor 6 (eIF6) is necessary for ribosome biogenesis and translation initiation. The eif6 gene is a single genetic locus highly conserved from yeast to humans indicating a tight regulation of its gene dosage. eIF6 haploinsufficiency protects mice from lymphomagenesis, and eIF6 is upregulated or amplified in some cancers, but a mechanistic study on the effects of eIF6 overexpression is still lacking. Taking advantage of genetic tractability of D. melanogaster, we characterized the first in vivo model of eIF6 upregulation. Drosophila eIF6 overexpression increases translation and results in a rough eye phenotype due to aberrant apoptosis. Mechanistically, eIF6 reshapes transcription and histone acetylation, disrupting the ecdysone network. This work is the first evidence of how increased translation generates a full transcriptional and hormonal dysregulation, providing new perspectives on the physiological relevance of the translational machinery in regulating gene expression and a model to screen drugs potentially useful to treat cells with altered eif6 gene dosage.Increases in ribosomal proteins and initiation factors are often observed in tumours and required during development. Haploinsufficient models have shown that such elevation is essential for tumour growth. Models with increased gene dosage of initiation factors, addressing the effects of their forced overexpression are lacking. The eukaryotic Initiation Factor 6 (eIF6) gene is amplified in some cancers and overexpressed in most, while it has been demonstrated that eIF6 haploinsufficiency protects mice from lymphomagenesis. eIF6 is necessary for ribosome biogenesis and efficient translation, and is present as a single gene in all animal species. Taking advantage of genetic tractability of Drosophila melanogaster, we generated an in vivo model of eIF6 upregulation, in order to assess the early effects of increased gene dosage of this initiation factor. eIF6 overexpression increases the general rate of translation, both in vivo and in vitro. Organ specific overexpression in the eye causes a rough phenotype. The increase of translation driven by eIF6 is accompanied by a complex transcriptional rewiring and a modulation of histone acetylation activity. Gene expression changes caused by eIF6 include a dominant upregulation of ribosome biogenesis, a shift in Programmed Cell Death (PCD) and inhibition of ecdysteroids biosynthesis. Administration of 20-HydroxyEcdysone or expression of p35 apoptotic modulator reverts some of the effects driven by high eIF6 levels. We conclude that the increased translation driven by high levels of eIF6 generates a transcriptional and hormonal rewiring that evidences the capability of the translational machinery to regulate specific gene expression and metabolism. In addition, our in vivo model could be useful to screen potential drugs to treat cells with altered eIF6 gene dosage.