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.

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.

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).