ABSTRACTS Undergraduate STUDENTS

3D Printing Visualization of the Cholera Toxin and Escherichia coli Heat-labile Toxin

Kayla Aquilante, Bongsup Cho

This project utilizes 3D printing to assist in the investigation of the structural and mechanistic differences between the cholera toxin (CT) and the heat-labile toxin (LT). Analysis of models reveals a significant structural difference in each toxin's A2 chain, which ultimately contributes to their differential toxicities. The use of both physical and digital models facilitate a deeper understanding of these molecules.

Exploring the Neuroprotective Effects of Cannabinoids on Oxygen-Glucose Deprived Neurons in an In-Vitro Model of Stroke

Bhavya Chatragadda, Emily Potts, Hang Ma, Navindra Seeram, Claudia Fallini

Stroke is a leading cause of long-term disability and decreased mobility worldwide. During stroke, neuronal damage results from both the initial oxygen-glucose deprivation and the increase in excitotoxicity and oxidative stress after restoration of blood flow, a phase called Ischemia-Reperfusion Injury (IRI). Reducing the impact of these processes could result in better outcomes for post-stroke patients by preventing excessive neuronal death. Many natural compounds derived from the Cannabis Sativa plant, have been tested for neuroprotective effects due to their potential antioxidant properties. We hypothesized that neurons treated with cannabinoids immediately after experiencing an acute ischemic event will have an overall decrease in cell death compared to untreated controls. To test this hypothesis, we have performed a compound screen of various natural products on iPSC derived human cortical neurons that have been exposed to 30 minutes of oxygen and glucose deprivation. Our results suggest that neurons treated with cannabinoids following an acute ischemic event experience less cell death. These findings could provide evidence that natural products can serve as therapeutics to prevent neuronal death in post-stroke patients.

Investigating the Role of CPNE2 in Chromatin Remodeling, Transcription, and Stress Granule Metabolism

Kristen Harder, Danielle Burge, Jaime M. Ross, Giuseppe Coppotelli

Copine 2 (CPNE2) is a poorly studied protein belonging to the “Copine" family, which are calcium-dependent, phospholipid binding proteins conserved across all species, including mammalians, where nine homologs (CPNE1-9) have been described. CPNE2 is highly expressed in the brain where we found it’s involved in the regulation of cognitive behavior, memory, and aggression in mice. Biochemical analysis of proteins interacting with CPNE2 suggests its involvement in RNA transcription, translation, and RNA stress granule formation. To understand more of the molecular function of CPNE2, we took advantage of a cell line that harbors 256 Lac operator (LacO) sequences and 96 tetracycline response elements with a minimal CMV promoter, which regulates the expression of CFP fused to the peroxisomal targeting signal SKL and 24 MS2 translational operators (MS2 repeats). Using this cell line, we investigated the role of CPNE2 in chromatin remodeling and transcription when tethered to LacO upon fusion with the Lac repressor (LacR) protein. Using live cell imaging, we investigated whether Cpne2 is recruited to the site of transcription and if calcium is needed for this process. We explored CPNE2 chromatin interaction by expressing GFP-CPNE2 in cells and used permeabilization to wash away unbound protein before microscopy analysis. Since our proteomic analysis of Cpne2 knock-out tissues revealed dysregulation of proteins involved in stress granule metabolism, we also investigated the involvement of CPNE2 in stress granule formation and clearance. We used sodium arsenite to induce stress granules in U2OS cells and determined whether RFP-CPNE2 is recruited to stress granules and if its overexpression affects stress granule clearance, using GFPG3BP1 as a marker. This study will help to understand the molecular function of CPNE2, thus elucidating its role in regulating memory and cognition, which could lead to treatments to ameliorate brain health and neurological disorders.

"Pinkalicious": Outer Membrane Vesicles Isolated from Pseudoalteromonas rubra Carry Prodiginine Antibiotics as Cargo

Margaret Hill, Arvie Masibag, Ololade Gbadebo, David Rowley

Astrangia poculata, the state coral of Rhode Island, is susceptible to infection by Vibrio coralliilyticus RE22, which is a common marine pathogen affecting various invertebrate species. Prior to the establishment of colonies, the settlement of coral larvae is influenced by crustose coralline algae (CCA) and their associated bacteria. Strains of Pseudoalteromonas rubra isolated from CCA have been shown to induce larval settlement of tropical corals. We hypothesize that P. rubra induces the settlement of A. poculata larvae by producing specialized metabolites packaged within membrane vesicles (MVs). In this study, we investigated MVs produced by Pseudoalteromonas rubra strains KB1 and CH007, which were isolated from CCA in the Roger Williams University Astrangia poculata coral culture tanks. MVs were isolated by ultracentrifugation, resuspended in HEPES buffer, and stored in a -80℃ freezer until further analysis. Frozen samples were extracted with organic solvents, dried on a rotary evaporator, and reconstituted in methanol. Samples were then adsorbed onto a Strata-X column, eluted with methanol and ethyl acetate, concentrated in vacuo, and reconstituted in either methanol or ethyl acetate at 1 mg/mL. Chemical analysis was completed using an LTQ-XL mass spectrometer equipped with a Phenomenex Kinetex 2.6 µm C18 (150 x 4.6 mm) LC column. We concluded that the MVs contained a family of antibacterial agents known as prodiginines. Prodiginines are a class of red-pigmented secondary metabolites that have a broad spectrum of biological activities, such as antimicrobial and algicidal activity, toxigenicity, and immunosuppressive properties. P. rubra OMVs would likely select for cargoes that exhibit antagonism towards competing strains. The future directions for this project will include further metabolomic analyses of P. rubra OMVs, with the intention of focusing on prodiginines as cargo.

Investigating the Molecular Mechanism of Action of the Sesquiterpene Lactone

Laurenobiolide

Oli Horyn, Kira Bernabe, Hannah Trautmann, Sierra Schmidt, Steven Gregory, Matthew Bertin, Kathryn M. Ramsey

Investigating the Molecular Mechanism of Action of the Sesquiterpene Lactone Laurenobiolide

Oli Horyn1, Kira Bernabe2, Hannah Trautmann2, Sierra Schmidt2, Steven Gregory2, Matthew Bertin3, Kathryn M. Ramsey2,3

1. Pharmacy Practice, University of Rhode Island, Kingston, RI

2. Cell and Molecular Biology, University of Rhode Island, Kingston, RI

3. Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI

With constantly evolving bacteria threatening the efficacy of antibiotics, the search for novel antimicrobials is imperative. Natural products historically have been used medicinally and have provided lead compounds for drug development. Laurenobiolide is a sesquiterpene lactone isolated from the North American tulip tree Liriodendron tulipifera. It has antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Using disc diffusion assays, we validated its activity on a methicillin-sensitive strain of S. aureus, found activity against Francisella tularensis, and observed no activity against Escherichia coli at the tested concentration. We also isolated S. aureus colonies that grew closer to the laurenobiolideimpregnated disc and confirmed that three isolates are laurenobiolide-resistant mutants. To investigate what genetic changes might lead to resistance, we used high-throughput sequencing to re-sequence the genomes of the laurenobiolide-resistant mutants and wild-type cells. This allowed us to identify what mutations are present in the laurenobiolide-resistant mutants but not the original laurenobiolide-sensitive cells. In all three resistant isolates, we found two mutations resulting in changes to coding sequences: (1) a frameshift mutation in a gene encoding a class I SAM-dependent methyltransferase leading to a premature truncation and (2) a single nucleotide change (SNP) in the rplU gene encoding the ribosomal protein bL21 that results in a change of amino acid 89 from an arginine to a proline. Given these mutations result in changes to proteins which may be essential for cellular function, we hypothesize one of these two mutations may cause laurenobiolide-resistance. Together, we validated that laurenobiolide exhibits antimicrobial activity on S. aureus, assessed laurenobiolide for antimicrobial activity against other bacteria, and identified two potentially resistance-causing mutations. Our work suggests that laurenobiolide may be developed as a novel, potentially broad-spectrum, antimicrobial and our ongoing work is expected to provide insight into how laurenobiolide exerts its effects at the molecular level.