Zebra mussels are an introduced species that has spread throughout much of the eastern United States and recently invaded Texas. These freshwater mussels cause ecological damage by reducing food available and outcompeting native clams. They cause significant economic damage by attaching to hard surfaces in the water such as pipes to factories and water treatment plants. Understanding where they might spread is an important step in controlling their invasion. Predicting their distribution can be challenging; however, several factors are indicative of where zebra mussels may spread (pH levels, temperature, calcium). Of these factors, calcium is currently viewed as the most significant. Zebra mussels needing calcium for general blood physiology, creating their calcium carbonate shells, and by developing larvae which have small shells. The working model is that zebra mussels will thrive in waters with calcium levels greater than 27mg/L, zebra mussel adults may survive but the larvae may not survive in calcium levels between 27-12 mg/L, and less than 12mg/L of calcium is too low for any stage of mussels to survive for an extended period. My projected look at survival of zebra mussels at varying concentrations of calcium in waters on zebra mussels. Differing calcium levels of 0, 5, 10, 20, and 30 mg/L in artificial pondwater were be used to determine at what levels of calcium zebra mussels are able to survive.

Cryptococcus neorformans is a fungal pathogen that mainly affects immunocompromised patients and is opportunistic as it invades the central nervous system. In the Wormley research lab, we are currently working with multiple genes that have been shown to be involved in lipid metabolism. C. neorformans. Using the TRACE procedure; Transient CRISPR-Cas9 coupled with Electroporation is hypothesized to be a reliable method in order to knock out genes in C. neoformans. This specific project we have been working on will lead to a knockout by using CRISPR methodology to create a Cryptococcus neoforman deletion construct for an associated gene. We are analyzing identified genes that have been found to be upregulated in C. neoformans, multiple of which have been shown to be involved in lipid metabolism and virulence. By characterizing the role of these genes and certain proteins this project aims to deepen the knowledge of the roles of lipids in pathogenesis and hopefully develop ways to combat infection of people with weakened immune systems. To further characterize the role of these genes in virulence we focus on the gene CNAG_00474, which was upregulated in C. neoformans in the presence of arachidonic acid. To achieve this we will generate a KO using the TRACE technique. The overall problem in this study is the implications these fungal proteins may have and the lack of understanding surrounding their involvement which is essential to research in order to create a pathway leading toward potential drug targets. The methodology of this project includes the amplifying promoter and end sequences from the unidentified protein to fuse the primer with sgRNA to create a construct ultimately. From here, amplifying the ‘arms’ of the target protein and the selection marker from a plasmid (in this case NAT) will use PCR to fuse the marker and arms together and create the deletion construct. Once we confirm the gene has been knocked out we will analyze its role in virulence by assessing phenotypic characteristics in vitro and in vivo. I am hoping in the near future to have the specific KO ready to be able to confirm it via PCR.

Marine environments are at risk of contamination from oil refinery effluents, major oil spills, and wastewater runoff. To identify and mitigate such risks, the EPA requires toxicity testing of marine effluents. The larval growth and survival (LGS) test, featuring either sheepshead minnows (SHMs) or inland silversides (INS), is currently used to screen marine effluents for acute toxicity; however, the use of fish larvae represents an animal welfare concern, especially in light of legislation calling for adherence to the 3Rs of animal research. The fish embryo toxicity (FET) test and mysid growth and survival (MGS) test may represent viable alternatives to LGS tests as the FET test uses fish embryos (which are thought to experience less pain than older fish), while the MGS test uses invertebrates. The objective of this study was to determine if the FET and/or the MGS tests produce similar results as the LGS. To accomplish this, INS LGS, SHM LGS, INS FET, SHM FET, and MGS tests were run using phenanthrene, an environmentally-relevant component of crude oil. Results revealed that the LC50 values obtained from the MGS and INS LGS tests were comparable and that both were significantly lower than that of the other test types, suggesting that the MGS test may be a viable replacement for the LGS tests. This was further substantiated when growth metrics were evaluated. In contrast, the LC50 values obtained from both FET tests were significantly higher than those of the other test types indicating a relative lack of sensitivity. However, when hatchability was included as a test metric, the sensitivity of the INS and SHM FET was enhanced indicating that the inclusion of hatch may improve FET test performance.

Due to our rapidly aging population, 6.5 million Americans currently have Alzheimer’s disease (AD), and this is predicted to increase to almost 14 million in the next 40 years. AD is more prevalent in western societies, and researchers suggest that this may be due to the typical Western diet. In contrast, AD prevalence is lower in Mediterranean regions, where a healthier diet could be a contributing factor. Therefore, this research examined the neuroprotective potential of a Mediterranean diet against AD pathologies and inflammation in mice. Our lab designed two experimental rodent diets, one that mimicked a typical Western-style diet, and another that mimicked a typical Mediterranean diet. We examined the lifelong effects of diet on biological markers of AD, including amyloid beta, a protein that aggregates together to form plaques in the AD brain, and pro-inflammatory cytokines, which are associated with increased inflammation. We hypothesized that the Mediterranean diet has the potential to mitigate these AD pathologies and therefore, could potentially be used as a future preventative strategy for AD.

Bacillus anthracis is the causative agent of anthrax. Previously, our lab identified the clpX gene as critical for virulence in B. anthracis. The ΔclpX mutant exhibited decreased cell wall integrity and increased susceptibility to cell-envelope active antibiotics. ClpX is one component of the intracellular caseinolytic protease ClpXP that degrades multiple proteins including transcriptional regulators. To understand changes in gene expression in ΔclpX, a microarray comparing WT and ΔclpX was conducted. This project focuses on msrA, an upregulated gene in ΔclpX. MsrA is an antioxidant enzyme that reduces methionine-S-sulfoxide to methionine but also impacts cell wall strength in S. aureus. This study will determine if loss of the msrA gene impacts antibiotic susceptibility. We hypothesized that since ΔmsrA is upregulated in ΔclpX, ΔmsrA would exhibit the opposite phenotype. Surprisingly, we find that ΔmsrA has significant growth inhibition in the presence of penicillin. However, we do not find susceptibility with other antibiotics, such as daptomycin, nor does it appear to be more susceptible to other clpX-related stress responses such as heat or acid stress. Future research will test ΔmsrA susceptibility to additional antimicrobials, such as the antimicrobial peptide LL-37 and the antibiotic vancomycin, as well as ΔmsrA virulence in vivo with the Galleria mellonella infection model. We will also complement ΔmsrA to confirm the phenotypes are due to loss of the msrA gene. This research is important as it aids our understanding of bacterial defenses and may provide new drug targets to help combat rising antibiotic resistance.