16 Feb 2018 Abstracts

Bioactive Fungal Screening to find Efficient Biomass-Degrading Enzymes

Uriel Vasquez-Rios '18

Traditional fuels such as coal and gasoline are limited and can produce harmful effects to the environment. Bioethanol from biomass (i.e., plant-based materials) degradation is a sustainable and renewable form of fuel that aims to reduce these effects. The fungal secretome, known as a pool of biomass degrading enzymes, can be utilized to discover industrial enzyme candidates useful for biofuel production. The overall purpose of our research is to pre-screen a pool of fungi collected from the Great Lakes, select candidates that can break down biomass (i.e., cellulose) efficiently, and characterize the biomass degrading enzymes. In this study, we first selected seven different fungal species, and grew them onto carboxymethyl cellulose (CMC) plates. A specific dye called Congo red was used to monitor the cellulose activity after either a five-day or twelve-day growth period. A clear zone present around the central fungal growth indicated the degradation of cellulose in the culture medium. Fungal activity was measured using a ratio of enzyme activity (outer ring) and the fungal growth (central circle). The enzyme activities of these strains were compared to Aspergillus niger, a known industrial fungal strain. Overall, there were four different species that demonstrated higher cellulase activity in comparison to A. niger in the CMC assay. Additionally, the secretome and cell lysate of A. niger was prepared to determine the source of the biomass-degrading enzymes through an enzymatic activity assay. Preparation of the secretome and lysate were obtained through ultra filtration and bead beating, respectively. Both methods resulted in acquiring a highly concentrated solution of each protein samples. It was determined that the biomass-degrading enzymes were secreted from the fungal cells rather than contained within the cell. Future directions include secretome preparation of the four identified fungal strains to verify their biomass hydrolysis capabilities.

Efficacy of Lentiviral Gene Therapy in Dogs and Mice with Hemophilia B

Rachel Spears '18

Hemophilia is a sex-linked genetic disorder that causes spontaneous bleeding and an inability for blood to clot in extreme cases. Hemophilia can be divided into two types, Hemophilia A and hemophilia B. Hemophilia B is caused by a deficiency of coagulation factor IX (FIX). Previous researchers have used gene therapy to treat people with severe hemophilia B by administering a dose of HIV-directed lentiviral vectors to target liver cells during transcription of miRNA to increase the efficacy of coagulation factor IX. The aim of this research was to repeat the treatment in a larger animal with hemophilia B (dogs) as well as evaluate the risk of

mutagenesis upon insertion of the lentiviral vector in mice that were tumor-prone. Three lentiviral vectors were inserted into three different dogs with severe hemophilia B and clotting time as well as overall health was evaluated. All dogs responded well to treatment, and experienced major reduction in both amount of spontaneous bleeding as well as clotting times of the bleeds. Effects of the lentiviral vector into tumor prone mice was evaluated by inserting the lentiviral vector into 27 mice, 9 of which were sacrificed after 3 months and 18 at 12 months. Once sacrificed, the mice livers were evaluated by lentiviral vector content and integration. Treated mice were found to not have increased evidence of genotoxicity, therefore making this therapy a viable treatment for severe hemophilia B, with further research needed to viably treat other forms of hemophilia.