Mercury (Hg) emitted in temperate and tropical regions can be transported to the Arctic where it is disproportionately deposited across the landscape. In aquatic systems, inorganic forms of Hg can be methylated to the toxic and bioaccumulative species, methylmercury (MeHg). In temperate zones, riparian spiders that specialize in consuming adult insects emerging from aquatic systems (e.g., Araneidae and Tetragnathidae) accumulate high concentrations of MeHg and have been used as sentinels of MeHg contamination. In addition, these taxa frequently accumulate concentrations of MeHg that may pose a risk to arachnivorous songbirds. Although these taxa are useful sentinels in risk assessment studies in the temperate zone, they are not present in the High Arctic. The purpose of the present study was to assess the potential of a generalist spider species, the Arctic wolf spider (Pardosa glacialis), to serve as a sentinel of Hg pollution in the Arctic. In summer 2022, we collected 1460 wolf spiders and 8090 emergent aquatic insects (Chironomidae) from six ponds in Northwest Greenland (centered around 76.5° N, 68.8° W). Spiders and insects were composited by body size and collection site. Hg concentrations for spiders and insects ranged from 230 - 1100 ng/g dry weight (dw) and 75 - 297 ng/g dw, respectively. Spider Hg concentrations were strongly correlated with insect Hg concentrations (R2 = 0.83), suggesting that wolf spiders can be used as sentinels of Hg contamination in Arctic lentic systems and had Hg concentrations that exceeded risk thresholds for arachnivorous songbirds.

Bacteriophages, the most abundant biological entities on Earth, specifically infect bacteria. These viruses initiate the lytic cycle, hijacking the cellular machinery of their bacterial hosts to replicate, which ultimately leads to the host's destruction. Phage therapy has shown promising results in treating antibiotic-resistant infections, though clinical trials are ongoing to fully establish its safety and efficacy. Identifying suitable phages is crucial in developing successful therapy due to the specificity of bacteriophage-host interactions.
Our study refined methods for isolating and studying bacteriophages against Enterobacter aerogenes, a critical ESKAPE pathogen contributing to antibiotic resistance. We evaluated two isolation techniques: the overnight enrichment assay and direct isolation via the whole plate spotting assay. Our comparison found an advantage of the direct isolation method—it not only matched the efficacy of the overnight enrichment but surpassed it by offering accelerated results and minimizing resource utilization. A key refinement for purification was the incorporation of calcium chloride into the soft agar, which markedly enhanced plaque clarity and visibility. Moreover, our exploration of DNA extraction techniques revealed the superiority of zinc chloride precipitation over commercial kits, with the former delivering higher DNA yield and purity.
We isolated three phages, K-1, BB-1, and M-1, effective against E. aerogenes. Noteworthy, phage BB-1 exhibited a rapid lytic cycle, clearing plates in under 10 hours. Future research will focus on examining their infectivity across Enterobacter strains, lysis of host cells, and absorption rates. We will also analyze their genome sequences to determine their novelty and potential for addressing antibiotic resistance.

Both Breast Cancer Gene 1 (BRCA1) and Partner and Localizer of BRCA 2 (PALB2) interact through their coiled-coil domains and are critical for proper functioning of DNA repair through homologous recombination. Individuals who carry pathogenic variants in either protein have an increased risk of developing breast cancer. Multiple variations of unknown significance (VUS) have been documented within the coiled-coil region of each protein, meaning a person carries a variation in either BRCA1 or PALB2 but there is not enough data to know the clinical significance of those variants. In this study, we analyzed the structural effects of five proline VUS within the binding region of BRCA1 and PALB2, three variants that have been discovered in patients and two variants that have not yet been discovered in patients. We used a known benign valine variant and a known pathogenic proline variant as our negative and positive controls, respectively. Because proline variants are known to disrupt protein structure, we hypothesize the proline variants will inhibit coiled-coil formation and, in turn, disrupt interaction between BRCA1 and PALB2. Each coiled-coil construct was generated in vitro and its structure analyzed using circular dichroism first individually and then in the presence of its wild-type binding partner. While all proline variants affected the structure of BRCA1, they had different degrees of impact depending on where they are located in the coiled-coil. This suggests that proline variants impact the ability of BRCA1 to form a coiled-coil in the presence of PALB2 and may be pathogenic. Notably, however, the BRCA1 proline variants seem to cause different structural changes from the pathogenic PALB2 proline variant. This prompts further research to determine the structural effects on the residue level and to correlate structural changes with functional changes.

The presence of Reactive Oxidative Species (ROS) in the brain have been linked to the etiology of Alzheimer’s disease and neurodegeneration. In this project, novel antioxidant Indole derivative drugs were tested on BV-2 microglial cells using RT-qPCR to assess their ability to activate antioxidant gene expression. Nuclear factor erythroid 2–related factor 2 (Nrf2) is a gene transcription factor that is activated by oxidative stress and binds to a sequence called the Antioxidant Response Element (ARE), a region upstream of the DNA promoter sequence. Nrf2 activates transcription of antioxidative genes. Based on theoretical docking studies, we hypothesize that the novel compounds will disrupt the interaction between Nrf2 and its inhibitor KEAP, releasing Nrf-2 and enabling it to translocate to the nucleus. The novel antioxidant drugs should either increase the transcription of Nrf2-activated genes or reduce overall levels of antioxidative stress within cells. We tested for antioxidant properties by measuring Hemeoxygenase-1 (HO-1) and Nrf2 mRNA levels in BV-2 cells in the presence of these compounds.

Alzheimer’s disease (AD) is a neurodegenerative disease that is the most common cause of dementia. There is currently no cure for AD, which means the best alternative is developing preventative strategies, such as adoption of the Mediterranean diet (MD), which has been shown to reduce risk of AD development and mortality. In comparison, a typical American diet has been shown to increase risk of AD development. Although the ability of dietary factors to alter the propensity of development of AD is well established, the mechanisms through this is mediated is unknown. One largely unexplored mechanism of dietary-induced AD prevalence is epigenetic modification to genes associated with AD. The most studied epigenetic modification, DNA methylation of cytosines, has been known to alter gene expression levels. Therefore, this study aims to determine whether dietary influences can induce epigenetic modifications, and subsequently modify expression of genes associated with AD. To do this, DNA was extracted from hippocampal tissue of mice on either a MD or TAD, as well as their offspring who were consuming a control diet. The offspring were included to determine if differential methylation patterns are heritable, thereby implicating transgenerational effects and predisposition to AD development or protection. An epigenetic array was used to identify loci that were differentially methylated between diets, and qPCR was used to determine if differential methylation resulted in significant differences in gene expression. No loci were found to be significantly differentially methylated (p-val<0.0001) with an effect size of 10% or more, nor differentially expressed upon qPCR analysis.

Estrogen receptor alpha (ERα) and BRCA1 play an important role in the development of breast cancer, and multiple pathways link these two proteins together. Previous studies have identified the ligand binding domain (LBD) of ERα and residues 1 through 258 of BRCA1 as important in the direct physical interaction between these two proteins. This study aimed to characterize the binding kinetics of this interaction in the presence and absence of 17β-estradiol (estrogen) with a shortened BRCA1 construct (residues 177-258); however, binding between ERα LBD and this BRCA1 construct could not be detected through fluorescence emission spectroscopy or isothermal titration calorimetry (ITC). Synthesizing ERα LBD presented challenges with low yield, so the purification protocol was refined to cool bacterial cultures at an OD600 of 0.2 during growth and add dithiothreitol during lysis for improved yield. A 24% decrease in fluorescence intensity upon addition of estrogen to ERα LBD confirmed the ligand-binding functionality of the protein. Additionally, Stern-Volmer studies verified that the estrogen binding site on ERα LBD is located in close vicinity to the tryptophan residues in the protein since fluorescence quenching was more efficient in the absence of estrogen. Finally, factors contributing to the absence of ERα-BRCA1 binding are discussed, including the length of the BRCA1 construct used or the potential necessity of an additional protein, BARD1.

As antibiotic resistance increases and antibiotic development dwindles, new antimicrobial agents are needed. Recent advances in nanoscale engineering have increased interest in metal oxide nanoparticles, particularly zinc oxide nanoparticles, as antimicrobial agents. Zinc oxide nanoparticles are particularly promising due to their broad-spectrum antibacterial activity and low production cost. Despite many studies demonstrating the effectiveness of zinc oxide nanoparticles, the antibacterial mechanism is still unknown. Previous work has implicated the role of reactive oxygen species such as hydrogen peroxide, physical damage of the cell envelope, and/or release of toxic Zn2+ ions as possible mechanisms of action. To evaluate the role of these proposed methods, we assessed the susceptibility of S. aureus mutant strains, ΔkatA and ΔmprF, to zinc oxide nanoparticles. These assays demonstrated that hydrogen peroxide and electrostatic interactions are not crucial for mediating zinc oxide nanoparticle toxicity. Instead, we find that a soluble factor accumulates in Mueller Hinton Broth over time that mediates toxicity and that removal of Zn2+ through chelation reverses this toxicity. Furthermore, we find that the physical separation of zinc oxide nanoparticles and bacterial cells using a semi-permeable membrane still allows for growth inhibition. We conclude that soluble Zn2+ is the primary mechanism by which zinc oxide nanoparticles mediate toxicity in Mueller Hinton Broth. Future work investigating how factors such as particle morphology (e.g., size, polarity, surface defects) and media contribute to Zn2+ dissolution could allow for the synthesis of zinc oxide nanoparticles that possess chemical and morphological properties best suited for antibacterial efficacy.

BRCA1 and PALB2 proteins suppress tumor formation by promoting homologous recombination when DNA damage has occurred. Mutations in BRCA1 and PALB2 are associated with a higher prevalence of breast and ovarian cancers. Phosphorylation of BRCA1 and PALB2 occurs upon DNA damage and is vital for maintaining genomic integrity. The molecular mechanism of how phosphorylation directs the activation of these proteins is unknown. It is established that phosphorylation of BRCA1 and PALB2 occurs in or near the coiled-coil regions of both proteins. The proteins use this domain to heterodimerize, so we hypothesize that the phosphorylation events could promote efficient BRCA1/PALB2 interactions. Our study aims to determine the effect of phosphorylation on the BRCA1/PALB2 binding affinity. The serine and threonine residues that are phosphorylated on BRCA1 or PALB2 were mutated to a glutamic acid to mimic phosphorylation. Glutamic acid carries a negative charge and thus mimics the negative charge added to the protein upon phosphorylation. We overexpressed and purified the protein using a bacterial expression system and measured their heterodimerization affinity with isothermal titration calorimetry (ITC). We will share ITC data suggesting phosphorylation of PALB2 does not affect its binding affinity to BRCA1. The phosophomimicking mutations in BRCA1 have also been generated, both individually and in tandem, and we will share results from these binding studies that are ongoing and hypotheses generated from our results regarding phosphorylation as an activation switch to control BRCA1/PALB2 interactions.

The spread of red imported fire ants (Solenopsis invicta; RIFA) has frequently been cited as a factor contributing to the decline of the Texas horned lizard (Phrynosoma cornutum; THL). Two hypotheses for this are: 1) direct lizard mortality due to RIFA, and 2) displacement of THL’s main food source, harvester ants (Pogonomyrmex spp.), by RIFA. A new third hypothesis suggests that the invasion of RIFA could be causing a decline in hatchling THL food resources, as their diet is mainly composed of small ant species. Many studies have attempted widespread treatment to eradicate RIFA; however, this could have unintended consequences for non-target ant species that THL depend on. Using a targeted application method, we sought to reduce RIFA populations over the summers of 2022 and 2023 at four sites located at Mason Mountain Wildlife Management in central Texas, a locality with an ongoing THL reintroduction program. At each site, hot dog baits were placed 5 meters apart in 10x10 grids. At treatment sites, one teaspoon of Amdro (a common ant poison) was applied to baits with RIFA thirty minutes after placement. After Amdro was applied, baits were left for 30 minutes and then collected. Treatments were repeated monthly May – August. The effects of each targeted poisoning were evaluated using pitfall traps and ant abundance measurements from baits. Results showed that treatment may have decreased RIFA abundance for both years and had variable effects on hatchling food abundance. Future studies will increase sample size to better detect potential treatment effect.

The Identification of Novel Genes Related to Iron Acquisition in Bacillus Anthracis Sterne

Jessica Guilhas, Kyle Gallegos, Julio Manceras, Mariah Green, Jacob Malmquist, Shauna M. McGillivray

Bacillus anthracis, the causative agent of anthrax, is a spore-forming, gram-positive bacterium. Its virulence mechanisms are of interest due to its potential use as a biological weapon and high lethality. For B. anthracis to survive and reproduce in a host, it must evade the host's immune response and acquire nutrients. One important nutrient B. anthracis must acquire is iron. Iron is a limiting nutrient in the host because it is usually found sequestered to hemoglobin or bound to host proteins such as transferrin. To acquire iron, pathogens must strip it from the host proteins. To find genes important for iron acquisition from hemoglobin, we screened genetic mutants created through transposon mutagenesis. Media was chelated to remove all divalent cations, including iron, and then hemoglobin was added as the sole iron source. The mutants that were unable to grow were chosen to be tested in a larger volume hemoglobin assay. We confirmed the phenotype of several mutants using this larger volume assay and we are working to confirm the site of transposon disruption via PCR. The mutants thus identified include a mutation in a dUTPase gene and an L-aspartate oxidase gene, neither of which has been previously linked to iron acquisition from hemoglobin. Future directions include making independent mutations and/or complement the disrupted genes to confirm the gene disruption is linked to loss of iron acquisition from hemoglobin. This study allows for a further understanding of how B. anthracis acquires iron and sheds new light on potentially novel virulence mechanisms.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the formation of amyloid beta (Aβ) plaques in the brain and is the seventh leading cause of death in the United States. Chronic inflammation and oxidative stress associated with AD leads to neuronal cell death. A cellular protective mechanism against oxidative stress involves the Nuclear factor erythroid 2-related factor (Nrf2) pathway. Nrf2 is responsive to the reactive oxygen species (ROS) produced when the cell is under oxidative stress, leading to its translocation into the nucleus where it activates transcription of genes that produce antioxidant enzymes like heme oxygenase-1 (HO-1). To study this pathway in neurons, our lab chose to use the mouse hippocampal HT-22 neuronal cell line. Our previous attempts to grow these cells in culture proved difficult, leading us to hypothesize that providing a growth-enhancing surface of collagen would provide a more stable surface in which to propagate these cells. Here we show that HT-22 cells grown on rat tail collagen provide a model system to investigate the Nrf2 pathway. We also demonstrate that HT-22 cells are viable on tissue culture plastics without the need for collagen.

Approximately 1 in 9 Americans over the age of 65 has Alzheimer’s disease (AD). As the size of this age group is expected to more than double by 2040, the AD prevalence is likewise predicted to increase rapidly. Two key risk factors for late-onset AD include poor diet and obesity. Therefore, long-term nutritional strategies could potentially reduce the development of hallmark AD biomarkers, such as amyloid beta (Aβ), later in adulthood. Researchers have found that diets extremely rich in saturated fats are associated with increased Aβ production in both the cortex and hippocampus of rodents. Conversely, plant-based Mediterranean diets (MD) that are plentiful in unsaturated fatty acids were shown to mitigate Aβ in rodents.

The relationship between diet and AD biomarkers has been explored in prior animal research, yet most studies utilize extremely high fat diets (40-60% kcal fat) or supplement with individual MD nutritional components. To address these limitations, we designed comprehensive, macronutrient-matched Mediterranean and typical American diets (TAD) that mimic human diets in Mediterranean regions and the U.S., respectively. C57BL/6J mice were weaned onto one of the two diets at postnatal day 21. Following 6 months of diet consumption, we found that the TAD increased soluble Aβ1-42 in the brain. Additionally, mice on the TAD had excess hepatic lipid deposition, which is a hallmark of insulin resistance and metabolic dysregulation, a comorbidity linked to AD risk.

Exploring EncT Efflux Pump Functionality and their Role in Lipid Signaling
Gabby Linares, Sawyer Diaz, Natalia Castro-Lopez, Floyd Wormley Jr.
Department of Biology, Texas Christian University

Cryptococcus neoformans, a fungal pathogen mainly affecting immunocompromised individuals, has sparked interest in lipid signaling research due to its role in pathogenesis. Eicosanoids, derived from fatty acids, are crucial in virulence and immune modulation; with C. neoformans lacking human enzyme homologs for eicosanoids biosynthesis, we want to identify the enzymes involved in the biosynthesis of cryptococcal eicosanoids and test their potential as antifungal targets. This project is focused on the EncT gene, encoding an efflux pump, which we observed to be upregulated in response to lipid precursors. Using CRISPR technology, we produced an EncT knockout (KO) strain and the corresponding reconstituted strain, aiming to discern shifts in virulence factors like melanin production, capsule formation, and urea production, among others, comparing the knockout, wild-type, and reconstituted strains and, subsequently, employing a mouse model of pulmonary cryptococcosis to delve deeper into virulence dynamics. Our initial results show early production of melanin EncT KO compared to the WT strain and no changes in the capsule formation or growth at 37°C.

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.