Fabian Lopez1,2, Cameron Bowers3, Giri Akkaraju1,3, Texas Christian University1, Department of Chemistry and Biochemistry2, Department of Biology3

Microglial cells are resident immune cells in the human brain that mediate the inflammatory response. The molecular hallmarks of Alzheimer’s Disease are neurofibrillary tangles and amyloid-B protein aggregates. In response to this buildup of these proteins, microglial cells release pro-inflammatory cytokines, such as TNF-alpha to recruit other microglia to this site of injury. However, when the microglia are unable to remove the waste, there is then a continuous cycle of cytokine secretion and microglia recruitment that leads to chronic inflammation. The NF-kB pathway is activated when molecules of bacterial cell walls, such as LPS, bind to toll-like receptor 4 (TLR4) in infected cells. This results in the translocation of NF-kB to the nucleus where it induces the expression of the TNF-alpha gene. In order to attenuate this response, our collaborators at P2D Biosciences and the Lab of Dr. Kayla Green at TCU have designed anti-inflammatory drugs. BV-2 cells are microglial mouse cells that are used as a model to test the efficacy of these drugs. The cytotoxicity of these drugs was first measured using an MTT assay to ensure that any observed reductions in secretions of cytokines such as TNF-alpha can be attributed to inhibition of inflammatory signaling pathways by the drug. An Enzyme-Linked Immunosorbent Assay (ELISA) was utilized to quantify and compare the levels of TNF-alpha in control and drug treated groups. The preliminary results suggest that Dr. Green’s drug, PK60, leads to a reduction in the levels of TNF-alpha secreted by BV-2 cells. This work serves as basis for employing techniques to investigate how upstream messengers of the NF-kB pathway are affected by PK60 to identify its mechanism of action.

BRCA1 and PALB2 are two proteins that bind to efficiently repair DNA damage through homologous recombination. Inability for these proteins to dimerize due to genetic variations can increase an individual’s risk of developing breast and ovarian cancer. Currently, most PALB2 genetic variants are classified as variants of unknown significance (VUSs) due to insufficient data to predict pathogenicity. In vivo methods to predict pathogenicity of these variants are time consuming and costly. As a result, we aimed to create a high-throughput and cell-free assay to test the effect of VUSs on the BRCA1-PALB2 binding interaction. Importantly, we wanted to recreate any relevant cellular conditions to obtain the most accurate data, and currently, the effect of PALB2 phosphorylation on the BRCA1-PALB2 binding interaction in vitro is unknown. To determine if phosphorylation affects the binding interaction, we mimic the phosphorylation states of PALB2 using site-directed mutagenesis and test their effect on BRCA1 binding using isothermal titration calorimetry. Our results indicate a surprising finding: PALB2 phosphorylation does not significantly alter the strength of the BRCA1-PALB2 binding interaction with minimized constructs in vitro. Thus, we hypothesize it is not critical to recreate the phosphorylation states of PALB2 when testing the effect of VUSs on the BRCA1-PALB2 binding interaction.

With the surge of multidrug resistant bacteria and increasing antibiotic resistance, there is a critical need for the development of new drug therapies. A new antimicrobial technique revolves around targeting virulence factors, which enable the bacterial pathogen to evade host immune defenses. Inhibitors that target pathogenicity hinder the capacity of the bacterium to cause an infection, thus allowing the host immune system to better clear the infection. In this study, we aim to inhibit the ClpXP protease, a highly conserved intracellular protease involved in virulence in different bacterial pathogens. Previous studies have shown that inhibition of ClpX completely attenuates virulence in Bacillus anthracis, rendering the pathogen more susceptible to cell envelope targeting antibiotics such as penicillin, daptomycin and LL-37. Computational modeling was performed and ten commercially available inhibitors with predicted activity against ClpX were identified, with ritanserin showing the most promise. In this study we explore the antimicrobial effects of ritanserin, a previously identified serotonin 2A receptor antagonist that underwent clinical trials as a potential treatment for schizophrenia and substance dependence. We hypothesized that if ritanserin inhibits ClpX in B. anthracis Sterne it should mimic the phenotype of the knockout clpX mutant, ΔclpX. We found that ritanserin increased WT Bacillus anthracis susceptibility to the cell envelope targeting antibiotics penicillin and daptomycin. Future studies will look at interactions host defenses such as antimicrobial peptides including LL-37. This demonstrates that ritanserin could be potentially repurposed as an antibacterial drug with the potential to be used by itself or in combination with antibiotics.

Hydrilla verticillata is an invasive aquatic plant found in freshwater systems throughout the United States. Invasions pose a threat to plants and animals that come into contact with the rapidly expanding hydrilla, and recreational activities such as fishing and boating are disrupted when densely packed mats of vegetative material form near the surface of the water. These invasions have historically been controlled using the herbicide fluridone, but resistant hydrilla populations have emerged with mutations in the pds gene associated with the production of phytoene desaturase. These resistant mutant plants are outwardly indistinguishable from the susceptible wildtype, and as such it can be challenging for aquatic systems managers to identify whether or not fluridone can be considered as an effective treatment option without some form of genetic testing. The existing standard process for identifying resistant mutants is a lengthy process that relies on amplifying and sequencing the pds gene. Our work has sought to utilize the process of double-mismatch allele-specific qPCR (DMAS-qPCR) to create a quicker and more cost-effective tool to aid in understanding the extent of fluridone resistant hydrilla. Having designed sets of primers specific to the mutations found within the pds gene, the next step is to apply this method to screening hydrilla samples from a variety of geographies in order to outline the spread of the resistant mutant populations.

Alzheimer’s Disease (AD), the most common form of Dementia, is a brain disorder that affects memory, cognition, and behavior. It currently affects 6.7 million Americans in the United States and interferes with daily life. Neuroinflammation in the brain is thought to worsen symptoms and drive the progression of the disease. Inflammation is mediated by the transcription factor NFkB, which typically leads to transcription of pro-inflammatory cytokines, including TNF-alpha and IL-1B. The transcription of these cytokines can lead to a cycle of chronic inflammation if left unregulated. In collaboration with P2D Biosciences and the Green Lab, we focused on testing compounds for their ability to reduce inflammation. Some of the compounds tested here have been shown to reduce cognitive defects in a mouse model of AD. In this study we are trying to understand the mechanism of action of these drugs. We are looking at the effect on the transcription factor NFkB.

The prevalence of antimicrobial-resistant bacteria is a rapidly growing public health crisis. This, combined with a decline in the development of novel antimicrobial therapies, makes the search for unique drug targets essential. Previous work from our lab has identified a promising antimicrobial drug target within Bacillus anthracis, the regulatory ATPase, ClpX. ClpX is essential for virulence in B. anthracis and critical for resistance to a host of cell envelope-targeting antimicrobials. ClpX works with ClpP to form a global protease that regulates a wide range of proteins, including transcriptional regulators. Previously, we conducted a microarray of a ΔclpX mutant and found 119 genes with altered expression. One such gene, msrB, has been studied for reactive oxygen species tolerance in other pathogens. This gene encodes for methionine sulfoxide reductase, an antioxidant enzyme that restores functionality to oxidized methionine residues. Increased msrB expression was seen with oxacillin exposure in S. aureus, indicating a potential connection between MsrB and cell wall-targeting antimicrobials. In B. anthracis Sterne, loss of msrB induces sensitivity to penicillin, but unlike ΔclpX, this phenotype is not seen with daptomycin or LL-37. This suggests that the role of msrB in antimicrobial tolerance may be limited to cell wall active antibiotics. Further experiments will include testing the ΔmsrB mutant with additional cell wall-specific antimicrobials (e.g., bacitracin and vancomycin). Our research provides additional information regarding the role of MsrB in the bacterial cell and its potential suitability as a pharmacological target to increase susceptibility to antibiotics.

B. anthracis is a gram-positive, spore-forming bacterial pathogen and the causative agent of the deadly disease, anthrax. This pathogen produces a lethal infection due to the potency of its virulence factors in inflicting harm upon and defending against their host. While anthrax toxin and capsule encoded in the B. anthracis plasmids are well-studied, there is minimal research into the over 5,000 chromosomal genes. To identify potential chromosomal virulence factors, a B. anthracis Sterne strain transposon mutant library containing thousands of randomly disrupted genomes was created and previously used to successfully screen for loss of virulence-associated phenotypes. In our current screen, we examined attenuation of mutants exposed to oxidative stress in the form of H2O2. ROS are released by innate immune response cells and destroy invading pathogens lacking adequate defense mechanisms. While there are some known antioxidant-encoding genes in B. anthracis, like the catalase gene, we predict there are others that may influence the bacteria’s susceptibility to ROS. To search for additional genes, we screened over 1,300 transposon mutants using H2O2 and selected mutants with growth attenuation compared to wild-type B. anthracis Sterne. Mutants with increased H2O2 susceptibility were further tested to confirm in-vitro phenotypes. Ultimately, we want to screen selected mutants in the G. mellonella invertebrate infection models to prioritize mutants with both in-vitro and in-vivo phenotypes. Our goal is to discover novel virulence factors while also developing validated methods and procedures to study B. anthracis pathogenesis.

Mercury (Hg) is released into the environment by coal-fired power plants, artisanal gold mines, and other human activities. Aquatic bacteria then convert the inorganic mercury into a highly toxic compound called methyl mercury. The methyl mercury builds up through bioaccumulation and biomagnification causing consumption bans for several species of fish in Texas. Dragonfly larvae can be used as bioindicators for methyl mercury contamination in aquatic ecosystems. The carnivorous diet of larvae leads to the bioaccumulation of measurable amounts of methyl mercury. Fort Worth ISD students have been working with TCU on the USGS citizen science - “The Dragonfly Mercury Project”. Dragonfly larvae are collected by students using dipnets at the Fort Worth Nature Center and Refuge along with National Parks across the United States. The larvae are placed in Ziploc bags with a label indicating the family, total length, date and location. Students wore gloves and followed a strict protocol to avoid contamination of samples. The samples are frozen and shipped with dry ice to a USGS lab for analysis. We report the data collected during 2018, 2020, 2021 and 2022 at the Fort Worth Nature Center and Refuge. We sampled three different sites on the refuge, Lake Worth (n=62, x̄=20.5ppb), Lotus Marsh(n=62, x̄=61.4ppb), and West Pasture Pond(n=66, x̄=34.4ppb).

Exploring the effects of a comprehensive Mediterranean diet verses a typical American diet on peripheral inflammation and the expression of inflammation-related genes in the dorsal hippocampus
Catherine Shoffner, Mary Skrabanek, Raleigh Robinson, Caleb Pryor, Morgan Bertrand, Vivienne Lacy, Paige Braden Kuhle, Gary Boehm, Michael Chumley
Approximately 72% of Americans are overweight or obese, partially due to the consumption of a Western diet (WD). The highly-processed WD is composed of simple carbohydrates, sugars, and saturated fats. The WD has been identified as a risk factor for Alzheimer’s disease (AD) due to the elevated levels of pro-inflammatory cytokines following long-term diet consumption. In contrast to the WD, the Mediterranean diet (MD) is a plant-based, mostly unsaturated fat diet. Research has shown that it is crucial to consume a balanced omega-6 to omega-3 ratio of 1:1 or 2:1, like that in the MD, as elevated ratios found in the WD lead to increased inflammation.
Previous studies generally utilize an extremely high-fat Western rodent diet that does not resemble that of the typical American. Thus, our lab designed two novel macronutrient-matched diets that mimic typical American or Mediterranean diets. In the current study, we examined the effects of the typical American diet (TAD) versus the MD in relation to pro-inflammatory cytokine production in serum and gene expression in the dorsal hippocampus of C57BL/6J mice. Following six months of TAD or MD consumption, the mice were treated with one intraperitoneal injection of lipopolysaccharide (LPS) or saline 4 hours prior to euthanasia. In comparison to the MD, mice consuming the TAD had increased expression and levels of pro-inflammatory cytokines in the dorsal hippocampus and serum, respectively.

In attempt to characterize the toxic effects of effluents discharged into surface waters, a previous study has shown crude oil contamination alters both mortality and hatching success among two model organisms: inland silversides and sheepshead minnows. Through toxicity testing it has become apparent that pollutants have the capacity to significantly alter growth and development of marine life. Specifically, it was found that exposure delayed the time of hatch or didn’t allow for hatch at all, and the unhatched embryos were less likely to survive. In addition, differences in both hatch and mortality were observed between the two organisms. Since the experimental conditions of the previous study were held constant for both groups the observed differences must be a result of a physiological difference, and a key distinction between the two species may lie in the differential use of the yolk sac. In the early stages of development marine organisms utilize the yolk sac as an internal source of energy prior to free feeding. It has been indicated that energy reserves and rate of depletions can differ between species. To determine the rate of yolk sac depletion in both inland silversides and sheepshead minnows, a total of 48 embryos per species were collected and raised to hatch. A subset of larvae at 24- to 96-hours post hatch were collected and the presence or absence of the yolk sac was determined. It was found that the yolk sac was depleted in 100% of the larvae 24-hours earlier for inland silversides as compared to sheepshead minnows. This observed difference shows a difference in the rates of energy reserve use and is indicative of a dissimilar response to external stressors, such as crude oil. These results may provide evidence of a mechanisms by which marine organisms experience differential hatch success and mortality when exposed to pollutants. Future research efforts might focus on the effect of yolk sac depletion as a key physiological distinction between species when outlining adverse effects of additional chemical exposures.

Oncorhynchus mykiss, commonly known as rainbow trout, exhibit partial migratory behavior, in which some individuals in a population will opt to migrate, whereas others do not. Consequently, there are two ecotypes of O. mykiss: the non-migratory rainbow trout (also known as residents) and the migratory steelhead (also known as migrants). Previous evidence generated from our lab demonstrated that various loci in the rainbow trout genome segregate between resident rainbow trout and migrant steelhead trout in the Sashin creek system of Alaska. A unique feature of the Sashin system is that a series of waterfalls separate the lake and stream, thereby inhibiting gene flow between the lake between migratory stream individuals and resident lake individuals. However, it is still unknown whether these same genetic markers also segregate between behaviors in other freshwater systems. Therefore, the goal of my research project is to use DMAS-qPCR to genotype known migrant individuals and known resident individuals from Little Sheep Creek, Oregon. This population is geographically separated from the Sashin Creek watershed and differs from Sashin in that both life histories can and do interbreed. From this project, I will be able to deduce 1) if genetic markers associated with life history development are shared across freshwater drainages and 2) to test if there is evidence of assortative mating (i.e., residents mating with residents and migrants mating with migrants) within the Little Sheep Creek system which would suggest genetic differences between life histories.

Mercury (Hg) is a global contaminant produced primarily by anthropogenic activities (i.e. coal-fired power plants, artisanal gold-mining operations) and is found in all freshwater systems. Primary producers (e.g., algae) and aquatic organisms that consume algae (e.g., emerging aquatic insects) are exposed to mercury through their diet. As adults, these emerging insects leave freshwater systems to reproduce, transferring both energy and Hg from their aquatic environment to the adjacent terrestrial environment. We assessed the emergence biomass of aquatic insects and insect–mediated Hg flux from 6 ponds in Northwest Greenland from July 1-30, 2022. Emergence biomass ranged from 0.09 to 176.91 mg/m2/day and insect-mediated Hg flux ranged from 0.009 ng/m2/day to 23.67 ng/m2/day across all ponds for the sampling period. This study suggests that small pongs in the High Arctic are important sources of both energy and contaminants to food webs in surrounding terrestrial ecosystems.

Alzheimer’s disease (AD) is ranked as the seventh leading cause of death in the US with over 6 million Americans currently diagnosed, and that number is projected to reach about 13 million by 2050. AD is currently believed to be caused by numerous factors ranging from genetics, lifestyle, and environmental conditions. The exact pathogenesis of AD remains uncertain, but the pathology of the disease includes the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles composed of the protein tau in the brain. These are two proteins are normally found in the brains of healthy individuals, but amyloid-beta peptides are often degraded under normal conditions, while tau plays a role in stabilizing our cell’s cytoskeletal structures. In Alzheimer’s however, these proteins are misfolded and accumulate, causing disruptions in cell signaling and neuronal death, therefore worsening the disease. Aβ plaques also activate microglial cells, which produce cytokines and induce inflammation. Cytokines are signaling molecules produced by immune cells that mediate inflammatory signaling. Activation of an inflammasome complex found in microglial cells, NLRP3, leads to the production of the cytokine IL-1β which has been implicated in Alzheimer’s due to its ability to induce and maintain this chronic cycle of inflammation, and possibly results in more amyloid-beta deposition. Our research looks into the mode of action of novel anti-inflammatory drugs and their potential to reduce inflammation at the level of the NLRP3 inflammasome as a mechanism to slow down the progression of AD.
 

Understanding the diversity and distribution of species on Earth is crucial in the face of contemporary threats to biodiversity, such as climate change and unsustainable economic practices. Unfortunately, the process of documenting and describing biodiversity often cannot keep pace with habitat loss and species extinction, especially in tropical regions where the number of undescribed and poorly known species is highest, and where biodiversity is most severely threatened. If this diversity is not documented, it will mean a loss of valuable understanding of the natural world and a failure to recognize species whose societal values remain undiscovered or underappreciated. This research will assess the extinction risk of selected fern species to understand their conservation status. The focus lies on understanding the classification, distribution, and conservation status of a group of species within the fern genus Elaphoglossum, the Elaphoglossum dendricola Clade, consisting of around 12 species distributed in the Tropical Andes, mostly at high altitudes (over 2400 m). This assessment aims to serve as a baseline for future conservation studies of this neotropical group of ferns.

Mercury contamination is of increasing concern. As the earth’s temperature continues to rise, it is vital to study the trends of MeHg absorption. Continuing to gather MeHg absorption data in the Northwest part of Greenland will help to grow our understanding of MeHg trends in Arctic territories. This study will increase the amount of data collected on MeHg levels, allowing a more accurate comparison between MeHg level patterns and species behavior, breeding success, and death rates in Arctic bird species (Chastel et al., 2022). Understanding how MeHg contamination affects health and prosperity is critical, not only for the environment and animals but people as well, as these birds are often part of the native Greenlander diet (Hong et al. 2012; Johansen et al. 2004). Temporal monitoring is also highly beneficial for evaluating the efficacy of policies aiming to reduce anthropogenic Hg emissions (AMAP 2021).

Due to widespread anthropogenic emissions and a global atmospheric cycle, mercury contaminates all aquatic ecosystems, including in the Arctic, at concentrations above pre-industrial baselines. Many seabirds nest in large colonies in the Arctic and are at elevated risk of mercury contamination due to their planktivorous and piscivorous diets and long lifespan. We investigated temporal trends of mercury contamination in five species of seabirds from northwest Greenland. Blood samples were collected regularly since 2010 from adult Atlantic puffins (Fracterula arctica), black guillemots (Cepphus grylle), black-legged kittiwakes (Rissa tridactyla), dovekies (Alle alle) and thick-billed murres (Uria lomvia). Samples were analyzed for total mercury using direct mercury analysis. All species had average blood mercury concentrations between 212 and 769 ng/g-wet weight, concentrations associated with a low risk for mercury toxicity. Individual black guillemots and thick-billed murres had blood mercury concentrations >1,000 ng/g wet weight, concentrations associated with moderate risk for mercury toxicity. Preliminary analyses suggest an overall increase in mercury concentrations in black-legged kittiwakes, dovekies and thick-billed murres over the study period. Comparable temporal studies in the Arctic have shown wide variation in mercury contamination trends. The results of the present study contribute to the understanding of regional mercury trends in the Arctic and efforts to assess the impact of the Minamata Convention.

As the number of antibiotics in development dwindles and antibiotic resistance continues to rise,
there is a need for novel, non-traditional antibiotics such as zinc oxide nanoparticles (ZnO NPs).
While the broad-spectrum antimicrobial properties are well established, the mechanism of action
is still unknown. Previous work has proposed that reactive oxygen species (ROS), toxic Zn2+ ions,
and electrostatic interactions with the cell envelope may be implicated in the mechanism. To
evaluate which of these mechanisms are involved, we characterized the physical and genetic
properties that confer resistance to ZnO NPs in three novel ZnO resistant strains of
Staphylococcus aureus (ZnOR). These strains possess comparable growth rates and are at least
four times more resistant than the parental strain against ZnO NPs acquired from multiple
sources. This suggests that all ZnO NPs, regardless of morphology, size, or method of synthesis
share a mechanism of action. We found that cell charge, measured by cytochrome c, was not
different between the parental and resistant strains, indicating that electrostatic interactions
with the membrane are not involved in the mechanism. Additionally, the ZnOR strains shared a
similar susceptibility to H2O2, a ROS commonly suggested to be generated by ZnO. We have also
found that internalization and physical contact with the bacterial envelope are not necessary for
ZnO mediated growth inhibition suggesting that ZnO produces a soluble species that is
responsible for the antibacterial action. Future work includes sequencing the genome of the
parental and ZnOR strains to identify mutations that led to gain of resistance.

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 the causative agent of the fatal disease anthrax, and its virulence is of great interest due to its potential as a biological weapon. B. anthracis causes disease by both escaping immune defenses and acquiring nutrients. A necessary nutrient that pathogens must acquire from its host is iron. To discover novel genes essential for iron acquisition, we screened transposon mutants in iron-deficient media with hemoglobin as the sole source of iron. We further prioritized the mutants discovered in our in vitro screen by assessing for attenuated virulence using our in vivo G. mellonella infection model. We found one mutant that has a disruption in the first gene of a two-gene operon containing putative dUTPase and aminopeptidase genes known as 9F12 Tn. Neither of these genes have been previously linked to iron acquisition. To confirm the role of the dUTPase gene in the observed 9F12 Tn phenotype, we created an independent insertional mutant in the dUTPase gene (dUTPase IM). We found that both of our mutants, 9F12 Tn and dUTPase IM, could not use hemoglobin as a source of iron. We also found that G. mellonella injected with 9F12 Tn and dUTPase IM had higher survival rates than those injected with the parent strain. Our results indicate that the dUTPase gene is necessary for iron-acquisition and virulence in B. anthracis. This study furthers our understanding of iron acquisition in a bacterial pathogen and increases our knowledge of how B. anthracis causes disease.