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.

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).

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.