Cryptococcus neoformans is an ubiquitous fungal pathogen that is detrimental for immunocompromised patients, leading to pneumonia and fatal meningoencephalitis. Fungal signaling lipids termed eicosanoids have been associated with increased virulence in this pathogen. Since C. neoformans lacks common enzymes associated with eicosanoid biosynthesis in humans, this pathway presents novel genes which could be used as potential drug targets. Our study focuses on EncT, a poorly characterized gene which encodes an efflux pump and is involved in the production of eicosanoids in C. neoformans. To evaluate the potential role of this gene in virulence, we used CRISPR technology to knock out (KO) the EncT gene, followed by screening for stable mutants and confirming the gene deletion via DNA amplification. After constructing the KO, we conducted in-vitro virulence assays of the KO strain and the wild-type strain (H99), including tests to assess sensitivity to temperature and changes in virulence factors including the production of melanin and capsule. These tests will help us characterize the potential role of the EncT gene in the virulence of this pathogen. Future directions include using a similar gene-editing method to generate an EncT reconstituted strain for use as an additional control in the in-vitro assays. Further, H99, the KO strain, and the reconstituted strain will be given to mice to evaluate the pathogenicity of the KO strain in an in-vivo model. Additionally, we will evaluate the presence of the fungi in the lung and the dissemination in other organs, and we will analyze the host immune response. By knocking out a gene involved in virulence-associated lipid production and characterizing the role of this gene in pathogenicity, this project will broaden the knowledge of the role of lipids in fungal pathogenesis and provide information that could potentially assist in developing therapies against this pathogen.

In a remarkable work of symbiosis, the gut microbiota coordinate with the brain to regulate multiple bodily functions, including those of the immune system, through bidirectional communication with the gut-brain axis. This symbiotic process has been shown to affect human health and disease pathology as certain inflammatory responses correlate with the composition and general disruption of the gut microbiome. To name a few, neurological disorders, gut-based inflammatory disorders, and cancer have been linked, in part, to dysfunction of the gut-brain axis. Previous literature on the gut-brain axis stems from in vivo and in vitro models, which have worked to understand the connection between the microbiome and disease pathology. Emerging evidence from these studies has continued to become more convincing regarding the importance of the bidirectional relationship in human health. In this review, evidence focusing on the intricate connections between the gut-brain axis and several inflammatory diseases, including irritable bowel syndrome, celiac disease, Crohn's disease, cancer, lupus, and Alzheimer’s disease, will be discussed. How this information can be utilized, including what has been or could be done in the clinic to improve the outcomes of patients with inflammatory-related diseases, will be highlighted so that continued advances in this newer aspect of medicine might lead to direct benefits for human health.

Bacillus anthracis is a gram-positive bacterial pathogen that causes the deadly infectious disease anthrax. B. anthracis contains over 5,000 chromosomal genes, and we believe there are unidentified chromosomal genes important for virulence. Our lab constructed a transposon mutant library with random disruptions in the B. anthracis Sterne genome to screen for novel virulence factors, and we have previously identified two virulence genes, clpX and yceGH, using this library. In this screen, we used hydrogen peroxide, a reactive oxygen species involved in innate immune defense, and screened around 1000 mutants. We obtained three mutants that were susceptible to hydrogen peroxide in vitro: 11F11, LV1, and LV2. To determine whether they also had phenotypes in vivo, we infected Galleria mellonella to study their virulence in an invertebrate animal infection model. LV2 showed reduced virulence in the in vivo survival assay, and all three mutants showed reduced virulence in the in vivo competition assay. I have determined the site of the transposon insertion in 11F11 and LV1, and the transposon has inserted in the genes for catalase and a collagenase-like protein, respectively. I am currently creating an independent insertional mutation in LV1 to confirm that the observed phenotypes are linked to the disruption of the collagenase-like protein. Future directions include creating a complementation plasmid for LV1 and determining the insertion site of LV2. The findings of this research could be used as potential therapeutic drug targets and will offer insight into the mechanisms that B. anthracis uses for its pathogenesis.

Alzheimer's Disease (AD) affects approximately 6.5 million Americans, and there is currently no cure. Prior research has shown that a key pathology of AD is amyloid beta, a protein that aggregates and form plaques in the brain, under pathological conditions. If amyloid beta is not cleared by the body, resultant plaques may disrupt proper cognitive and neuronal function. As the liver plays a crucial role clearing amyloid beta, liver damage may jeopardize the efficacy of the liver to clear amyloid beta in the periphery of the body, enabling it to reach the brain.

One way liver function can be disrupted is through diet, specifically the Western diet (WD), which has been shown to cause non-alcoholic fatty liver disease (NAFLD) and inflammation, both of which are associated with AD. A WD is classified as one that contains high amounts of refined sugars and saturated fats derived from animals. Conversely, the Mediterranean Diet (MD), a largely plant-based diet, contains high amounts of monounsaturated fatty acids and polyunsaturated fatty acids. These dietary factors have been shown to decrease inflammation and increase antioxidant effects, further protecting the brain from AD pathology. Therefore, we hypothesize that the MD could protect the liver and be used as a potential prevention strategy for NAFLD and AD.

The current study examined the effects of WD and MD on the relationship between the liver and the brain in wild type mice. During tissue collection, livers were taken and histologically analyzed. The livers from each experimental group were processed, stained, and evaluated for their overall composition.

BRCA1 and BARD1 are proteins involved in the repression of genes associated with increased risk for breast and ovarian cancers. This is accomplished through ubiquitination of H2A and subsequent changes in chromatin compaction. BRCA1 and BARD1 form an E3 ligase (BCBD complex), and mutations affecting the enzymatic functions of this complex can predispose women to these cancers. The model organism C. elegans contains orthologs of these proteins, BRC-1 and BRD-1, which makes it a useful organism for studies of protein function; however, little is known about the mechanism of ubiquitination in C. elegans as compared to humans. This project used nucleosome assays to provide more insight on the ubiquitination of H2A by the BCBD complex in C. elegans. The objectives of this project included characterizing the interaction of the BCBD complex with H2A and identifying a specific lysine target in C. elegans. The conserved lysine targets were mutated out of H2A and nucleosome assays were performed to identify potential reductions in ubiquitination activity. In addition, we hypothesized that enzyme-substrate interactions, specifically between H2A and BRD-1 in C. elegans, are important in directing ubiquitin to the target site. Amino acid residues in BRD-1 thought to be important for these interactions were mutated out, and assays were performed to assess changes in ubiquitination activity. The H2A nucleosome assays showed that the mutations of conserved lysines in the H2A N-terminus and C-terminus in C. elegans did not significantly reduce ubiquitination activity, and a definitive target could not be identified. However, the BRD-1 assays identified amino acid residues in C. elegans that participate in directing the ubiquitination process. Further studies are needed to determine if C. elegans has any preferential lysine targets at a non-conserved residue or if it is truly nonspecific in its activity. Currently, mass spectrometry analysis is being performed as a complementary method to attempt to pinpoint the location of lysine ubiquitination.