• A Role for Taurine in Food Sensitivities in Fish

      Larkin, Mary Elizabeth; Place, Allen R.; 0000-0002-9421-0943 (2018)
      As the aquaculture industry expands to feed an ever-growing world population, it seeks to develop more low-cost, environmentally sustainable feed ingredients. Fish meal replacement by plant sources of protein, including those derived from soybeans, wheat, and peas may have unintended effects. Certain fish species have reacted poorly to particular ingredients, including components of soy and pea protein. We present here evidence for potential negative health effects, including possible inflammation, elicited by wheat gluten incorporation into the feed of Cobia (Rachycentron canadum). European sea bass (Dicentrarchus labrax), on the contrary, seem to well tolerate this ingredient. We sought to evaluate the ability of taurine, a known immunomodulator, to alleviate inflammation in cases of adverse effects caused by particular plant ingredients in feed. We saw evidence for this in the cobia study, and though our study did not induce any inflammation in sea bass, we detected dramatically increased levels of taurine levels in the plasma of fish consuming a diet containing 4% wheat gluten. In another study, supplemental taurine in European sea bass shifted spectral sensitivity to a longer wavelength, though there were no apparent anatomical differences in the retina between the un-supplemented and supplemented groups. We observed changes to the microbiome induced by dietary wheat gluten, and in a separate study, taurine. Wheat gluten addition to the diet greatly increased the number of predominant orders represented in the intestinal microbiota. Taurine caused less of a shift, but interestingly, the predominant orders were very uniform throughout the sections of the intestines of the taurine-fed fish. In two different studies, we attempted to characterize a dietary taurine-dependent zebrafish that was incapable of endogenous taurine synthesis. However, both of our potential strains turned out to be producing a wild-type CSAD (cysteine sulfinic acid decarboxylase) protein even in the presence of early termination codons in the csad gene. We also observed the expression of two smaller sizes of CSAD, ~53 and ~55 kDa, in addition to the previously described ~59 kDa protein. The two smaller sizes appear to be produced early in development and are not detectable by 3.5 weeks post-fertilization.
    • Discovery and Analysis of Patients with Monogenic Diabetes in Multiple Cohorts to Guide Future Diagnosis

      Kleinberger, Jeffrey; Pollin, Toni; 0000-0003-0521-9848 (2017)
      Monogenic diabetes is hyperglycemia caused by a variant in a single gene, and it accounts for approximately 1-2% of all diabetes cases. A genetic diagnosis of monogenic diabetes is important because the most common gene etiologies can be effectively managed with treatment regimens other than first line treatments for either type 1 (T1D) or type 2 diabetes (T2D). However, monogenic diabetes can have a similar clinical presentation to either T1D or T2D, leading to clinical misdiagnosis of monogenic diabetes. The goal of this dissertation was to evaluate approaches for identifying patients with monogenic diabetes and a method for functionally testing monogenic diabetes variants to potentially improve diagnosis and treatment of these patients. Monogenic diabetes genetic testing was performed on 488 samples from the Treatment Options for type 2 Diabetes in Adolescents and Youth (TODAY) clinical trial. A total of 4.5% (22/488) of individuals were determined to have pathogenic or likely pathogenic variants. Comparison of clinical characteristics of patients with and without monogenic diabetes discovered statistically, but not clinically, significant lower BMI Z-score, higher fasting glucose, and lower fasting insulin in patients with monogenic diabetes. Treatment outcomes from the TODAY trial showed that most patients with HNF4A monogenic diabetes variants failed treatment therapies rapidly, while none of the patients with GCK monogenic diabetes variants failed treatment. In the Personalized Diabetes Medicine Program (PDMP), an implementation study for screening, diagnosis, and return of results for monogenic diabetes, 1,734 participants were screened for monogenic diabetes at four diverse study sites. Of the 138 eligible participants that underwent monogenic diabetes genetic testing, 14 had pathogenic or likely pathogenic monogenic diabetes variants. PDMP patients with monogenic diabetes had a diverse range of ages, races/ethnicities, and previous treatment regimens. Finally, a zebrafish model of hnf1a-knockdown and rescue with HNF1A monogenic diabetes variants was evaluated to determine that the model could not accurately identify established damaging HNF1A genetic variants. The results from these studies have demonstrated the variable presentations of patients with monogenic diabetes as well as the challenges and potential of assessing the function of HNF1A variants using an in vivo model.
    • Molecular level studies of the impact of poly (oxonorbornenes) on D. rerio. embryos

      Klutts, Jennifer N.; Rosenzweig, Zeev (2019)
      Poly (oxonorbornenes) (PONs) are amphiphilic cationic polymers that possess antimicrobial properties. Cationic polymers are proposed as an alternative to antimicrobial peptides and it is important to assess their impact on organisms. The two side chains of PONs that are responsible for these properties are a hydrophobic alkyl and a charged amine. In this study, we investigated how changing the amine/alkyl ratio and polymer length affects the activity of PONs on the model vertebrate organism, D. rerio. (zebrafish). Zebrafish embryo toxicity test were used to elucidate the LC50 of PONs. Whole-mount immunofluorescence with caspase-3 was used to analyze apoptotic cells. We hypothesize that PONs would interact with the cell membranes of embryos to induce toxicity and the level of toxicity would depend on the molecular structure of PONs. Our results indicate that increasing the hydrophobicity and polymer length decreases the viability of the embryos and number apoptotic cells in the embryos.
    • Opposite Roles of Zebrafish Galectins in In Vitro Attachment and Infection by the Infectious Hematopoietic Necrosis Virus (IHNV)

      Abernathy, Kelsey; Vasta, Gerardo R.; 0000-0001-9218-2611 (2021)
      IHNV is a rhabdovirus with a high mortality rate that has major economic impacts on the salmonid fisheries and aquaculture industry. The virus uses cell surface fibronectin as a receptor for attachment and infection, but the exact mechanism remains unknown. Previously published work in our lab and preliminary data revealed that β-galactoside-binding lectins, known as galectins, interact directly with the IHNV envelope glycoprotein to either promote or inhibit viral attachment and infection of fish epithelial cells. The zebrafish tandem-repeat galectin 9 isoform 1 (Drgal9-L1) displays two carbohydrate recognition domains (CRD) joined by a linker peptide that are similar but not identical in binding specificity. The goal of this study was to explore the mechanism of Drgal9-L1-mediated enhancement of IHNV attachment to epithelial cells (EPC cell line). We showed that Drgal9-L1 crosslinks IHNV to cell surface glycans in a carbohydrate-dependent and -specific manner. We determined that crosslinking was dependent on two functional CRDs through the development of a C-terminal mutant protein that did not enhance IHNV attachment or infection of EPC cells. Drgal9-L1 crosslinks IHNV to fibronectin on the cell surface, enhancing viral attachment, in a carbohydrate -dependent and -specific manner. In addition, Drgal9-L1 binds to alternative ligands, β1-integrin and CD147, to increase IHNV attachment. Double antibody inhibition and siRNA knockdown of fibronectin and β1-integrin in EPC significantly reduced Drgal9-L1 mediated attachment of IHNV. We also investigated the protective role of epidermal mucus glycans for preventing Drgal9-L1 mediated viral attachment to the epithelium. All three galectin classes were detected in the zebrafish epidermal mucus, and Drgal9-L1 as well as Drgal1-L2 were found to bind to mucus glycans in a carbohydrate-specific manner. In a plaque assay, mucus coating of the cell monolayer reduced the number of IHNV plaques on the EPC cells in a concentration and volume-dependent manner and annulled the Drgal9-L1 enhancement of viral attachment and infectivity. Finally, we identified an alternative mechanism of Drgal1-L2 antiviral protective activity, as binding of Drgal1-L2 to surface glycosylated receptors or mucus glycans significantly inhibits IHNV attachment. This research has wide-ranging applications for aquaculture disease management, vaccine development, and a general model of galectin-modulated viral attachment.
    • Transglutaminase-Mediated Bone Formation in Zebrafish

      Deasey, Stephanie Catherine; Nurminskaya, Maria (2013)
      The integrity of the human skeleton is maintained by a delicate balance of bone deposition and resorption. Disruption of this balance results in diseases such as sclerosteosis or osteoporosis, which are characterized by high or low bone mass respectively. Further, the skeleton is prone to injuries such as fractures and breaks throughout the human life-span. It has therefore become critical to understand the underlying mechanisms of bone formation and homeostasis in order to better target and treat such ailments. Two mammalian enzyme transglutaminases, TG2b and FXIIIa, which catalyze the formation of protein-protein cross-links, have been associated with bone mineralization in vitro. However, mouse single knockouts of these enzymes show no skeletal phenotype. In this study we demonstrate functional and transcriptional compensation for the loss of TG2 in various tissues of TG2 knockout mice; specifically, we demonstrate a compensation mechanism in the skeleton. To overcome this complication we utilize the zebrafish (danio rerio) model system to examine the role of transglutaminases in vivo. Zebrafish have become an invaluable model to the study of developmental processes due to several unique characteristics such as, transparency during early development, short gestation time and a remarkable regeneration capability. We characterized the zebrafish transglutaminase gene family and identified thirteen TG genes. Of these thirteen genes, eleven were homologous to one of three mammalian transglutaminases, TG1, TG2, or FXIIIa, and two were specific to zebrafish. We show that transglutaminase activity promotes proper bone mineralization in both developing vertebrae and regenerating fin bones. Further, we show evidence for transglutaminases functioning in bone mineralization by promoting collagen type I deposition and activating canonical beta-catenin signaling during bone regeneration. Importantly, these studies settled the previous discrepancy between in vitro studies and in vivo mouse studies on the role of TGs in osteo-chondrogenic differentiation by demonstrating a complex compensation mechanism in mammalian tissue. This identification of TGs in bone mineralization identifies a novel therapeutic target for various bone pathologies, such as bone-like tissue transformation in heterotropic ossification seen in musculoskeletal trauma, spinal cord injury and combat wounds.