There are numerous known chemicals, present in surface waters, which may pose a significant risk to the health of fish living in aquatic environments. These chemicals can impair a variety of physiological process and to date, screening assays for evaluating acute, chronic, reproductive toxicity, and endocrine disruption have been well developed. However, screening assays capable of identify chemicals that can adversely impact immune function have yet to be developed. Developing such assays is necessary given that immune system disruption can increase the incidence of disease and death. Thus, the purpose of this study was to develop and validate a fish-based neutrophil migration assay that can be utilized to rapidly screen chemical for immunotoxicity. The specific objective of this study was to develop a neutrophil migration assay featuring adult fathead minnows (a commonly-used toxicity testing model organism). Specifically, this study sought to optimize methods for two key steps of the neutrophil migration assay – tail injury and depigmentation of fish tails for neutrophil visualization. Three tail injury methods were evaluated including partial amputation, a tail nick, and a biopsy punch. The methods of depigmentation evaluated were H2O2/KOH treatment alone, H2O2/KOH and acetone, and H2O2/KOH treatment in combination with acetone and FlyClear solution 1.1 (Triton X, THEED, and urea). Results showing which of these methods is best suited for neutrophil migration assays featuring adult fathead minnows will be presented.

Oxidative stress is an imbalance of reactive oxygen species (ROS) and antioxidant defenses resulting in cell damage and chronic inflammation. It contributes to many pathologies including neurodegenerative disorders, cardiovascular disease, diabetes, and cancer. Macrophages and microglia are phagocytic immune cells that destroy pathogens while releasing inflammatory mediators, such as pro-inflammatory cytokines and ROS. While inflammation is initially a protective mechanism, chronic inflammation is damaging to tissues. To counter oxidative stress, cells express nuclear factor-erythroid 2-related factor (Nrf2) to mitigate excess ROS production. Nrf2 is a transcription factor that promotes the expression of numerous antioxidant enzymes. Our study targets the expression and activation of Nrf2 in cells treated with L2, a compound created by Dr. Kayla Green (TCU Chemistry). Our lab is attempting to determine the molecular mechanism in which L2 may protect phagocytic cells from oxidative stress, and if this mechanism involves the Nrf2 pathway. This research could provide preliminary evidence for the efficacy of this compound as a treatment option for diseases involving oxidative stress.

With increasing global industrialization and subsequent pollution, there are mounting concerns regarding the presence and impacts of reproductive endocrine disrupting chemicals (REDCs), including environmental estrogens. These concerns have led to new international regulations (i.e. REACH) which require that chemicals be screened for endocrine disrupting activity. A variety of in vivo and in vitro screening currently exist; however, the in vivo methods are time-intensive and expensive and the in vitro methods may fail to detect estrogenic compounds with unique modes of action. Thus, there is a need for in vivo estrogen screen assays that are quick and inexpensive. The objective of this study is to validate the newly-developed Rapid Estrogen ACTivity In Vivo (REACTIV) Assay as a reliable approach for the detection of chemicals with estrogenic activity. This assay employs Japanese Medaka (Oryzias latipes) that have been genetically modified to co-express green fluorescent protein and choriogenin (an egg precursor protein). In the assay, the transgenic medaka embryos are exposed to a chemical of interest for 24 hours after hatch and then imaged under a fluorescent microscope. To validate the performance of the assay, tests were performed using two chemicals with known estrogenic activity (i.e., bisphenol A, estradiol) and two inert chemicals (i.e., saccharin and cefuroxime). Results showed that larvae exposed to the estrogenic compound experienced dose-dependent increases in liver fluorescence, while those exposed to the inert chemicals did not. Overall, these results indicate that the REACTIV assay produces predicable results and thus, may be appropriate for use as a standardized estrogen screen method.

BRCA1 is a gene found in humans that, when mutated, has been linked to breast and ovarian cancer. A homolog version of this gene, known as brc-1, exists in an organism called the Caenorhabditis elegans. This is a species of nematode worm that has the potential to be used as a model organism to study this homolog gene that is associated with human breast cancer. Previous studies with C. elegans have shown links between the brc-1 gene and DNA damage responses, cytochrome p450, or cyp, transcription levels, and ratios of male phenotype worms. This project focused on studying whether these brc-1 functions are dictated by the enzymatic activity of the protein made by this gene. To measure these phenotypes, we used a strain of C. elegans with a brc-1 mutation engineered to lack enzymatic activity of the BRCA1 protein toward nucleosomes. In order to determine how this lack of enzymatic activity affects brc-1 functions, we measured levels of reactive oxygen species (serving as a proxy for DNA damage), numbers of male offspring, and cyp levels in the mutant and wild-type C. elegans. Our initial results indicate the effects of enzymatic activity towards nucleosomes on the aforementioned phenotypes.

Staphylococcus aureus is the causative agent of many skin infections and the leading cause of death due to infectious disease in the United States. Additionally, S. aureus is known to rapidly gain antibiotic resistance, as seen with methicillin resistant Staphylococcus aureus (MRSA). Zinc oxide (ZnO), a nontraditional antibiotic, demonstrates antimicrobial action against S. aureus. While the exact mechanism of ZnO antibacterial action is currently unknown, production of reactive oxygen species (ROS) is a commonly proposed mechanism. We find that S. aureus ΔkatA, a mutant susceptible to hydrogen peroxide (H2O2) due to a deletion in the catalase gene, exhibits comparable growth to wild type S. aureus in ZnO. This suggests that production of H2O2 is not vital to the antimicrobial action of ZnO. To further test this, we generated a ZnO resistant mutant (ZnOR) that demonstrates less susceptibility to ZnO. We find that the ZnOR mutant demonstrates comparable growth to wild type S. aureus in H2O2, making H2O2 production an unlikely toxicity mechanism of ZnO. To evaluate the role of ROS besides H2O2, susceptibility of ZnOR and wild type S. aureus to two other ROS, bleach and paraquat was evaluated. We are currently investigating whether N-Acetyl-Cysteine (NAC), a compound that stimulates production of antioxidants and is protective against a wide range of ROS, protects S. aureus from ZnO mediated toxicity. Our data suggests that ROS formation is not the dominant mechanism of antimicrobial action by ZnO and future studies should focus on other potential mechanisms of action.

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.

Alzheimer’s Disease is a neurodegenerative disease characterized by cognitive, functional, and neuronal loss. Its core pathology includes beta-amyloid protein plaque formation, neurofibrillary tangles of tau protein, and loss of microglial cell function, all of which may be facilitated or exacerbated by a prolonged neuroinflammatory response. The inflammatory signaling pathway culminates in the activation of transcription factor NF-κB, which then goes on to activate the expression of cytokines and other signaling molecules such as TNFα. One of the points of regulation for this pathway is the constitutive binding of the IκBα protein to NF-κB that prevents NF-κB from entering the nucleus. However, when the appropriate stimulus triggers the pathway, a downstream effect is the phosphorylation of IκBα by the IκB kinase, and its subsequent degradation which then releases NF-κB for translocation into the nucleus.
This project aims to elucidate the mechanism of action of novel anti-inflammatory drugs (provided by P2D Biosciences company). Previous in vivo studies with the compound have shown a reduction in inflammation and improved cognition, but the drug’s exact point of interference in the pathway remains unclear. Therefore, this project aims to assess if the drug reduces inflammation by reducing IκBα degradation, thus preventing NF-κB from being able to turn on cytokine expression.
BV-2 mouse microglial cells were exposed to the drugs, followed by exposure to LPS for various time intervals, then harvested and lysed. A Western blot procedure was performed on the lysates to visualize the amount of IκBα present, then those bands were quantified to compare against control cells that were not incubated with the drug. It follows then, that if the drugs’ mechanism of action is inhibition of NF-κB release into the nucleus, then there will be increased amounts of IκBα in the treatment cells compared to the control cells as IκBα degradation is prevented.

According to Rita Charon, founder of the developing field of narrative medicine, "medicine practiced with narrative competence, called narrative medicine, is proposed as a model for humane and effective medical practice," which "offers fresh opportunities for respectful, empathetic, and nourishing medical care" (Charon, 2001). Narrative medicine is composed of three key practices: close reading, reflective writing, and active listening. Developing each of these skills, Sharon proposes, can foster compassion and empathy in medical providers. The demonstration of these practices has been shown to "facilitate an authentic partnership by building empathy and trust," as well as "promote physician well-being and prevent burnout" (Khawand-Azoulai, et. al. 2022, Stumbar, S. E et. al. 2020). Medical education currently is striving to incorporate humanistic training to develop a holistic approach to patient care, but narrative medicine training has yet to be extensively explored in undergraduate pre-health education (Pentiado, J. A. et. al., 2016, Barron, L., 2017). Narrative competence, defined by Charon as "the ability to acknowledge, absorb, interpret, and act on the stories and plights of others," has been proposed as a pre-requisite to developing good patient-care skills (Charon, 2001, Baron, L., 2017). The incorporation of humanistic training for undergraduate pre-medical students in the form of narrative medicine practices can prepare future professional school students to begin developing a patient-centered perspective of healthcare. The undergraduate years have the "potential to shape the kinds of caregivers we want for our patients, for friends, for our families, and for ourselves" (Barron, L. 2017). It is therefore important to gain an understanding of how medical humanities education can impact undergraduate students because foundational knowledge of these concepts prepares students for later development of humane medical practice in professional school. The three avenues of narrative medicine training that I have chosen to analyze include a narrative medicine workshop series, a group of pre-health students called the Illness Narrative Listening Project that gathers regularly to intentionally listen to patients stories, and lastly, my own reflective writing from experiences in medical contexts. Data is collected through a mixed methods approach, gleaning insight through both qualitative and quantitative research methods, in the form of interviews and surveys respectively, will illuminate the complexities of the research question. The goal of the workshops and listening project is to teach these practices to undergraduate students in order to explore if the same benefits seen in clinical practice and medical education could be demonstrated in undergraduate pre-health education.

Alzheimer’s disease (AD) currently afflicts well over six million people in the United States, and this number is projected to increase exponentially in the coming years. While much remains to be understood about the causes and pathogenesis of AD, two potential risk factors are chronic insufficient sleep and long-term consumption of an unhealthy diet. Both of these lifestyle factors are often studied separately, and evidence suggests that each has negative impacts on brain health and cognitive function, perhaps due to increases in inflammation, which itself is associated with increased anxiety and cognitive dysfunction. The current study investigated the combined effects of long-term consumption of a typical American-style diet (TAD) and six weeks of chronic sleep restriction on locomotor activity and anxiety-like behavior in male and female wild-type mice not otherwise predisposed to disease pathology. Female mice that underwent sleep restriction and consumed the TAD displayed greater anxiety-like behavior compared to mice that the TAD and did not undergo sleep restriction. This difference was not observed in male mice. Furthermore, male mice that underwent chronic sleep restriction displayed greater locomotor activity compared to controls. These differences were not observed in females. Given the prevalence of AD and the projected rise in AD cases, understanding how controllable lifestyle or environmental factors can increase AD risk is essential. Importantly, as AD is more prevalent in women compared to men, it is imperative that research efforts utilize male and female animals seek to understand the mechanisms driving this phenomenon.

There is an oxidative stress component to a wide range of neurobiological diseases. In Alzheimer’s disease (AD), secondary brain injury is associated with an imbalance between oxidant and antioxidant agents. This imbalance contributes to the pathophysiology of AD through the oxidation of macromolecules, destabilization of neuronal cells, and generation of ROS that upregulates synthesis and deposition of p-tau and Amyloid-β (Aβ). The expression of antioxidant defense enzymes can decrease damaging reactive oxygen species, so some efforts to alleviate secondary injury focus on this mechanism of reducing oxidative stress. One pathway that is activated in response to oxidative stress is the Nrf-2/ ARE pathway. Under stress conditions, the protein sensor for oxidation levels Keap1 that is bound to Nrf2 is oxidized, and Nrf2 levels are stabilized and subsequently increased in the cell. The Nrf2 transcription factor then translocates into the nucleus and binds to the antioxidant response element (ARE) promoter to turn on the expression of downstream antioxidant genes. The genes that are expressed include heme-oxygenase (HO-1) and NADPH quinine oxidoreductase 1 (NQO1). These antioxidants can then regulate the redox balance in the internal environment and reduce oxidative stress. The goal of my research is to design an assay to measure Nrf2 activation, so we can test drugs shown to reduce oxidative stress in vitro.

Alzheimer’s disease (AD), currently the seventh leading cause of death in the United States, is a neurodegenerative disease characterized by amyloid beta (Aβ) plaques and chronic inflammation in the brain. Microglial cells, which act as the immune cells of the central nervous system (CNS), function in response to Aβ by secreting pro-inflammatory cytokines and reactive oxygen species (ROS). Microglial activation is a healthy response in the brain, but chronic activation of these cells and thus chronic secretion of neurotoxic factors creates a cyclic process that leads to neuronal cell death. In order to protect against oxidative stress, cells activate the nuclear factor erythroid 2-related factor (Nrf2) pathway. Nrf2 is a transcription factor that regulates the expression of antioxidant enzymes, which can protect the cell from ROS. Here we focus on the therapeutic potential of cannabidiol (CBD) to mitigate oxidative stress in both microglial and peripheral macrophage cell lines. We show that CBD can activate the Nrf2 pathway and thus increases the expression of several antioxidant proteins such as Heme oxygenase-1 (HO-1). This research is significant because it could provide evidence for the use of CBD as a potential therapy in AD patients.

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.

The Molding Melanin Magic mentorship program through TCU Pre-Health is geared to impact minority female student populations at the Texas Academy of Biomedical Sciences (TABS) in Fort Worth. The program provides small group mentorship as high school students are paired with a college student in their area of interest. Along with mentorship, workshops are utilized as a method of increasing confidence, exposure, and overall knowledge about college and STEM careers. By coupling workshops and mentorship, the Molding Melanin Magic program seeks to encourage mentees to serve as mentors along their educational journey, and apply for college and professional school to pursue a career in STEM.

Spiders are sentinel species, organisms that serve to map the bioavailable fraction of contaminants in an ecosystem by retaining their contaminants in their tissues. For example, spiders in the families Tetragnathidae and Araneidae are frequently used as sentinels of mercury contamination of aquatic ecosystems. Spiders are frequently preserved in alcohol prior to contaminant analysis but the impact of contamination on mercury concentrations in spiders has not been assessed. The objective of the present study was to determine the effects of different preservation methods on mercury concentrations in tissues of spiders in the families Tetragnathidae and Araneidae. The spiders were collected along water sources using nets and gloved hands. The Tetragnathids were collected from grassy terrain or a bridge overhanging the water of Lake Weatherford. The araneids were collected from a boat dock overhanging Eagle Mountain Lake. On site, each spider was placed into its respective bottle of varying ethanol or Ziplock's for freezing. Individual spiders were placed into one of three different concentrations of ethanol (100%, 95% , 70%) or frozen. Following about two months of preservation, the spiders were dried and run through the DMA-80 collecting the data for data analysis. Data xxxx *insert conclusion info*

Role of ClpX in the stress response and virulence of Bacillus anthracis: protease or chaperone?
Lillian Wilson, Vuong Do, S.M McGillivray
Department of Biology, Texas Christian University

Anthrax is a lethal infectious disease caused by the bacterial pathogen Bacillus anthracis. Our lab studies the virulence and antibiotic resistance of B. anthracis and we have identified a chromosomal gene clpX, as an important virulence factor, as its loss increases susceptibility to cell-envelope targeting antibiotics such as penicillin, daptomycin, and the antimicrobial peptide LL-37. ClpX is an ATPase that can act autonomously as a chaperone, or with a proteolytic core, ClpP, to degrade proteins. To investigate the mechanism ClpX uses, a plasmid pclpXI264E was designed with a mutation in clpX (I264E) that prevents ClpP binding and inhibits the formation of the ClpXP protease but does not disrupt the chaperone activity of ClpX. We used this to create 4 strains in the unencapsulated Sterne strain: wild-type and ∆clpX containing the empty inducible plasmid pUTE657, complementation plasmid with the non-mutated clpX gene (∆clpX + pclpX), and the mutated plasmid (∆clpX + pclpXI264E). Prior research done on these strains confirmed that ClpX relies on protease activity in antimicrobial stress; however, our goal was to assess its response in other environmental stressors such as acid stress, heat stress, and its virulence in vivo with the Galleria mellonella infection model. We find that that the protease activity of ClpX is important for all of these stresses. These results build on our earlier understanding and demonstrate that formation of the ClpXP protease is critical and any future development of drugs targeting the ClpX system should focus on protease formation rather than chaperone activity.

This research is focused on gaining a better understanding of PKC-epsilon a calcium-dependent protein kinase involved in a wide range of cellular functions including cell proliferation, survival, and apoptosis. The interest in PKC-epsilon derives from the discovery of a de novo mutation in the PKC-epsilon gene in patients suffering from SHORT syndrome. This syndrome is a debilitating disorder characterized by short stature, hyperextensibility, ocular depression, Rieger anomaly, and teething decay. The project involved recapitulating the naturally occurring de novo mutation in vitro as well as determining if other mutations in PKC-epsilon could cause similar disease-state phenotypes. Using a technique known as Site Directed Mutagenesis mutations were introduced into the PKC-epsilon gene and the effects of these mutations on the protein expression were assessed. This mutational analysis will help identify the regions of PKC-epsilon that are vital for its function. This will help elucidate the effect of the same mutations in patients and could help predict the severity of disease. Obtaining a clearer picture of the different regions of the PKC-epsilon protein allows for future studies to focus on successfully fixing these regions when they become damaged and could therefore be used to help patients with SHORT syndrome.

N-terminal acetylation is essential for the stability, activity, and targeting of proteins in eukaryotes. However, most eukaryotic proteins are not acetylated when expressed in bacteria. Therefore, it is of practical significance to control N-terminal acetylation of recombinant proteins in bacteria. RimJ is an N-terminal acetyltransferase (NAT) known to acetylate many recombinant proteins with a narrow substrate specificity in E. coli. This project is aimed to increase the applicability of RimJ for the N-terminal acetylation of a broad range of recombinant proteins.
Based on the AlphaFold-predicted structure of E. coli RimJ, we predicted that six amino acids (Y35, E46, R49, Y106, Y170, and L171) may recognize substrate proteins in the active site. We created RimJ variants, in which one or two of these amino acids are changed to alanine, a small neutral amino acid, so that the active site becomes larger to accommodate substrate proteins containing bigger N-terminal amino acid residues. The RimJ variants were created using site directed mutagenesis, confirmed by DNA sequencing, and co-expressed with Z domain mutants that were not acetylated by the wildtype RimJ. The Z domain mutants were isolated by immobilized metal ion affinity chromatography and analyzed by mass spectrometry for their N-terminal acetylation patterns.