Alzheimer’s disease (AD) is often associated with chronic inflammation and cognitive dysfunction. In studying how AD-like pathologies change and affect learning and memory, our lab aims to optimize an object location memory (OLM) testing paradigm in mice. Briefly, a mouse is placed into an arena with two identical objects for a training session. Four hours later, one of the objects is moved to a novel location, and the mouse is placed back into the arena for the testing session. Because mice exhibit a preference for novelty, memory is assessed as the amount of time the mouse spends exploring the moved object divided by the total time spent exploring both objects. Our goal is to identify testing parameters that make this task both accurate and efficient for our lab’s use, as we will add this learning paradigm to a battery of behavioral tests to be used in future experiments. In the current study, the OLM protocol will be performed twice according to two different experimental timelines that test the effects of adding an additional training session to the original protocol.

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.

Our project focused on the conservation of activity of the protein BRD1 in C. elegans. C. elegans is a strong model organism for our study because BRD-1 is the worm ortholog to BARD1 in humans. Specifically, our focus is on its function as an enzyme to attach ubiquitin to the H2A tail of nucleosomes. We studied a structural mutation of BRD-1 that we predicted would interfere with its ability to bind its substrate, the nucleosome. We hypothesized that BRD-1 is bound to the nucleosome at this mutation site based on prior research in the human protein. Therefore, we integrated mutations found in humans into the DNA that codes for C. elegans BRD-1. A typical mutagenesis protocol was used to implement the mutations and then we expressed the proteins in E. coli cells. After that, nucleosomes were reconstituted by dialysis, and enzyme activity was assessed using a ubiquitination assay. These assays showed that BRD-1 in C. elegans does bind the nucleosome demonstrating conservation of the BARD1 function. Determining that function is conserved allowed us to determine that C. elegans is an appropriate organism to test mutations found in humans. This research has future clinical potential due to the ability to test mutations encountered in humans using a model organism and can aid with clinical treatment plans to help avoid the development of cancer.

Many anti-inflammatory drugs are currently in use to treat neuroinflammation in the brain which can result from Alzheimer's disease, Parkinson's disease, traumatic brain injury, and more. In collaboration with a company, P2Dbiosciences, we are testing drugs that can modulate the function of inflammatory cytokines such as TNF-alpha, with the goal of reducing neuroinflammation and thus benefiting people suffering from the neurodegeneration and cognitive decline associated with neuroinflammation. We hypothesize these drugs work by inhibiting the signaling associated with inflammatory cytokines.
Two different assays were developed to identify the mechanism of action of these cytokine modulating anti-inflammatory drugs. BV2 cells in culture were used for these assays to model how the drug affects mouse microglial cells (immune cells resident in the brain). The first assay uses a luciferase reporter gene to determine if NF-kB promoter activity is disrupted when cells are treated with drug. The second assay uses quantitative RT-PCR (qPCR) to measure changes in TNF-alpha mRNA levels when cells are treated with drug. Levels of TNF-alpha mRNA were also quantified over a period of time following drug treatment to determine whether the degradation time of the TNF-alpha mRNA was affected by treatment.

Invasive species, such as the Red Imported Fire Ant (Solenopsis invicta, hereafter, RIFA), can negatively impact native species via predation and modifying prey behavior. RIFA exist in two colony types, monogyne (single queen) and polygyne (multiple queens), and polygyne colonies are known to contain higher densities of fire ants than monogyne colonies. Texas horned lizard (Phrynosoma cornutum) eggs and hatchlings are suspected prey of RIFA’s foraging and aggressive behaviors. In this study, we collected fire ants from Karnes City and Kenedy to determine if Texas horned lizard density is lower around polygyne colonies. We collected and sequenced 30 ants, of which 20 were RIFA. Counter to our expectations, there seemed to be no correlation between RIFA colony type and Texas horned lizard density. Furthermore, we found evidence that monogyne and polgyne colonies were coexisting. In future studies, we think larger sampling sizes and determining ratios of polygyne to monogyne colonies within the same area would be useful for further testing the hypothesis that colony type may affect horned lizard density.

Emerging evidence suggests that the immune system is vulnerable to disruption in response to a wide variety of chemical contaminants; thus, there is a need to test chemicals for immunotoxicity. To understand how chemicals impact the ability of the immune system to ward off infection, a model system featuring fathead minnows (FHM, a common toxicological model) infected with Yersinia ruckeri (a bacteria) has been used. Using this model system, the impacts of chemicals on immune system function can be evaluated via pathogen resistance challenges, where a fish is infected with a pathogen and their ability to defend against the pathogen and survive infection is determined. However, the use of Y. ruckeri is unfavorable given that fish must be injected with Y. ruckeri to develop an infection, a process that is time consuming and inconsistent with natural routes of exposure. Thus, the goal of this project was to develop a new host-pathogen system for FHMs by identifying a pathogen that induces infection via immersion. To do this, the ability of three bacterial pathogens, Flavobacterium columnare, Aeromonas sobria and Aeromonas allosaccharophila, to infect FHMs via immersion was evaluated. Results of this study revealed that of the three bacterium evaluated, only F. columnare was capable of inducing an infection via immersion in FHMs and infection was only successful following a fin-clip procedure, in which a small portion of the caudal fin is removed. Overall, this result establishes the potential for a FHM-F. columnare model system for future use in immunotoxicity testing.

Nickel is the most prominent heavy metal in the effluents associated with crude oil extraction and production. Given that these effluents are released into the ocean, investigating the toxicity of nickel on marine life is pertinent. A current method for evaluating the toxicity of oil effluents is the sheepshead minnow larval growth and survival (LGS) test, which exposes larval fish to varying concentrations effluents or associated single chemicals over a 7-day period. However, current legislation, like the Frank E. Lautenberg Act, requires that animal testing be refined whenever possible to enhance animal welfare. The fish embryo toxicity (FET) test, which investigates chemical toxicity using fish embryos over a 7-day exposure period, is a potential alternative method capable of meeting legislative needs related to animal welfare. The objective of this study was to determine if a sheepshead minnow FET test is a viable replacement for the sheepshead minnow LGS test. To accomplish this, the results of sheepshead minnow LGS and FET tests using nickel were compared. The results of this study show that the LGS test is more sensitive than the FET test. In addition, evidence suggests that it may be possible to improve the sensitivity of the FET test by including sublethal metrics as FET test endpoints.

Bacillus anthracis is a bacterial pathogen that causes the often lethal disease anthrax. This research aims to characterize the role of potential virulence genes in Bacillus anthracis. Virulence is a pathogen’s ability to damage the host. Studying virulence allows us to understand infection mechanisms and develop novel ways to target pathogens. Previous work identified a collection of potential virulence mutants (Franks et al, 2014) each containing a genetic disruption that renders a gene non-functional. These mutants were pulled out in initial screenings but were never characterized further. We confirmed that one mutant, TN2, also exhibits decreased virulence in a Galleria mellonella survival assay. We know that TN2 has a disruption in a promoter region that we hypothesize controls two genes: a putative BNR repeat domain protein (TN2A) and a glycosyl-like 2 transferase family protein (TN2B). For my project, I attempted insertional mutagenesis to inactivate these genes with the goal of confirming that the genes are linked to virulence, rather than unintended mutations elsewhere in the genome. After successfully creating insertional mutant 2B, through the disruption of the TN2B gene, I am working to further characterize the mutant to determine its role in immune evasion. Specifically, I will compare the ability of the wild-type and mutants to survive exposure to various antimicrobial defenses conserved in humans and waxworms. This research could help identify a novel bacterial virulence factor and its potential mechanisms of action thus expanding our understanding of bacterial pathogenesis.

The gram-positive bacterium, Bacillus anthracis, is responsible for the deadly disease Anthrax. B. anthracis is dangerous due to virulence factors, or defenses the bacteria uses to infect a host. We hope to better understand how this bacterium interacts with its hosts by studying the genes necessary for virulence. Bacterial mutants, which have a change in their genetic sequence, sometimes show reduced ability to cause disease in a host. Studying these mutants helps us understand the bacteria’s infection method. Previously our lab created a library of mutants using a technique called transposon mutagenesis and then screened these transposon mutants for phenotypes linked to decreased virulence. This resulted in the identification of 11 transposon mutants that were less effective at causing disease in the nematode Caenorhabditis elegans (Franks et al.). While all 11 mutants could be interesting for further characterization, it is necessary to prioritize them as this is still too many to study. In this project, we tested these mutants using a second infection model, the caterpillar Galleria mellonella. G. mellonella is an ideal model due to its optimal size for injection, conserved innate immune defenses, and previous success as an infection model for B. anthracis (Malmquist et al.). We found that only one of these 11 mutants, TN2, had reduced virulence in both C. elegans and G. mellonella. Future research will focus on confirming the genetic change in this mutant and determining the mechanism by which it contributes to infection. This could lead to new antibiotic targets in the future.

Mercury (Hg) is released into the environment by coal-burning powerplants and artisanal gold mines. Aquatic bacteria then convert the inorganic mercury into highly toxic methyl mercury. Turtles acquire mercury through their diet, and it bioaccumulates throughout their long lifetime. Toenail clippings can be used to determine Hg concentrations in turtles. Toenail samples were collected from Trachemys scripta elegans (red-eared sliders) in the Brazos River near Granbury and the Clear Fork of the Trinity River as it flows through Fort Worth. All toenails were dried in a 60℃ oven and put into a direct Hg analyzer that uses thermal decomposition, gold amalgamation, and atomic absorption spectrometry to determine total Hg. Toenails from the Brazos river had significantly more Hg on average than those in the Clear Fork, 658.302µg/kg and 400.146µg/kg respectively. The results were unexpected as the Brazos river near Granbury is considered less polluted than the Clear Fork of the Trinity, which is supported by observations of insect larvae such as hellgrammites which were common in the Brazos but absent in the Clear Fork of the Trinity. Our hypothesis is that red-eared sliders in the Brazos are feeding at a higher trophic level than those in the Clear Fork. Fecal samples and a lack of invertebrate prey in the Clear Fork suggest red-eared sliders primarily feed on algae. In the Brazos river we observed several species of insect larvae underneath rocks and hypothesize that the red-eared sliders are feeding on this abundant food source. Mercury is known to biomagnify and therefore red-eared sliders in the Brazos are likely ingesting more mercury than those in the Clear Fork.

Alzheimer’s Disease (AD) and Traumatic Brain Injuries (TBI) are global societal problems affecting millions of people and costing billions of dollars per year.1,6 Hallmarks of AD include memory loss, cognitive decline, depression, and confusion due to unchecked inflammation in the brain caused by the overproduction of pro-inflammatory cytokines by the immune system.1,9,11 TBI occurs when a sudden trauma damages brain cells, which activate the immune response potentially leading to chronic inflammation and a multitude of symptoms affecting cognitive, somatic, and emotional processes.3,4,12 There is currently no cure for AD, nor is there an effective treatment for chronic inflammation caused by TBI. P2D Bioscience® has manufactured a series of drugs successfully targeting inflammation in a 3XTgAD mouse model.21 To understand the cellular mechanism of the novel drugs, we used SDS-PAGE electrophoresis and Western Blot analysis to investigate protein levels within the NFB pathway, which modulates inflammation. We monitored the inhibitor of NFb, IB, to determine whether the drugs were blocking the phosphorylation and degradation of IkBa and subsequently blocking the pro-inflammatory effects of activated NFB. We show that the drug is blocking the degradation of IB, and therefore the pro-inflammatory genes associated with the NFB pathway are not being transcribed. Increasing our understanding of the cellular mechanism of action is imperative for the progression of drug development because it can be used to evaluate potential side effects.

Alzheimer’s Disease (AD) is a neurodegenerative disease that primarily affects elderly populations. AD engenders memory loss and cognitive decline, and its prevalence is rapidly growing. It is estimated that 14 million Americans will have AD by the year 2050. Therefore, it is imperative for researchers to examine the underlying biological mechanisms responsible for AD. Previous research has demonstrated that chronic inflammation is linked to the hallmark AD pathology, amyloid beta (Aβ). Aβ is a protein that disrupts neuronal communication and increases the production of effector proteins called pro-inflammatory cytokines. Microglia function like immune cells in the brain, and when they are activated by inflammatory triggers, such as Aβ, they secrete pro-inflammatory cytokines. Although cytokine release is initially a healthy response, excess cytokine production is harmful to the brain and exacerbates AD pathologies. Prior research has demonstrated that pro-inflammatory cytokines are upregulated in the serum of AD patients. Therefore, cytokines are a crucial target for AD therapeutics.

The current experiment will examine the temporal inflammatory response of microglial cells following lipopolysaccharide (LPS) insult. LPS is a component of common bacteria and can induce inflammation in microglial cells. We will treat cells with several different concentrations of LPS and assess cytokine production at several different timepoints. To do this, we will collect cell supernatant (secretions) and measure multiple cytokines using an ultrasensitive electrochemiluminscent assay. Data collected from these experiments will be used in many future studies of potential therapeutics and dietary supplements. In fact, data from these experiments will be used by current and future departmental honors students to determine the optimal treatments and times for their experiments. This project is incredibly relevant because AD is currently the 6th leading cause of death in the United States. Data collected will help us pinpoint proper testing procedures for therapeutic compounds that are developed.

Cells use diverse mechanisms to prevent DNA damage and tumor formation. Two tumor suppressors employed in this effort are the focus of our study: breast cancer type 1 susceptibility protein (BRCA1) and BRCA-1-associated RING Domain protein 1 (BARD1). These two proteins form a complex that suppresses the generation of estrogen-derived free radicals. Inherited mutations in BRCA1 or BARD1 are associated with an increased risk of developing breast or ovarian cancer in humans. The model organism Caenorhabditis elegans possesses the orthologs BRC-1 and BRD-1 which can be readily mutated, offering an attractive model to study biochemical functions. However, it is unknown if BRC-1/BRD-1 also regulates the transcription of estrogen metabolism (cyp) genes to control the production of free radicals as noted for the human homologs. Utilizing gene expression analysis and estrogen exposure assays, this study demonstrates that BRC-1/BRD-1 has a conserved function of regulating cyp genes in C. elegans. However, our data also shows that BRC-1 and BRD-1 do not necessarily protect DNA from free radical damage upon estrogen exposure, despite its proven inhibition of cyp genes expression. Further investigation is required to determine the function of these cyp gene homologs in C. elegans. Our findings of this additional conserved function of the BRCA1/BARD1 homologs in C. elegans further validate its use as a model organism to better understand the myriad ways BRCA1/BARD1 protects the genome.

BRCA1 and p53 have been proven to interact in tumor suppressor pathways for hereditary breast and ovarian cancer. Finding the physical binding location associated with this interplay is important in assessing cancer risk and determining molecular details of the interaction. This project aimed to identify the protein binding region of p53 with the intrinsically disordered region of BRCA1. We cloned select regions of human BRCA1 and p53 protein into E. coli bacteria, then harvested and purified the proteins. A pull-down assay was performed to test binding affinity between a segment of p53 and two different length BRCA1 constructs. The assay showed that neither the construct that contained BRCA1 amino acids between 772-1126 nor the construct with amino acids between 896-1190 interacted with p53. This indicates that these amino acids alone are not sufficient for binding of p53 and BRCA1. Our results could also indicate that a third-party binding mediator is required in vivo. This information expands upon our knowledge of the p53 and BRCA1 binding interaction and can be used in a clinical setting to evaluate risk associated with mutations in the experimental regions.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease associated with old age and marked by deficits in memory and learning skills. AD pathology is characterized by amyloid-beta (AB) accumulation, which leads to plaque formation and ultimately neuronal death. Additionally, AB activates microglial cells, which function as an immune cell in the brain. Microglial cells secrete proteins that induce inflammation, known as pro-inflammatory cytokines. The chronic activation of pro-inflammatory cytokines engenders neuroinflammation and oxidative stress, which then further exacerbates AD pathologies. This project aims to study the effectiveness of cannabidiol (CBD) as a potential treatment for AD, due to its known anti-inflammatory properties. We will measure the inflammatory response of cultured BV2 immortalized mouse microglial cells following lipopolysaccharide (LPS) treatment. We will then include a CBD treatment to study its therapeutic capabilities in reducing inflammation. We hypothesize that treatment with CBD will decrease the pro-inflammatory cytokines TNF-alpha and IL-6 induced by LPS stimulation. We will perform enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the cytokine levels.The overall goal of the research is to demonstrate the capacity of CBD to minimize the immunological mechanisms that drive AD pathologies. Our research will contribute to the understanding of the link between the immune system and central nervous system in AD development. AD is the sixth leading cause of death in America, but the availability of therapies is limited. CBD represents a natural and possible effective therapy for those suffering from Alzheimer’s disease, and our research will contribute to determining its efficacy.

Effect of Season, Body Size, and Sex on the Mercury Concentrations of Orb-Weaving Spiders
Cale Perry, Garrett Helburn, Olivia Eberwein, Madeline Hannappel, Matthew Chumchal, and Ray Drenner
Mercury (Hg) is an anthropogenic contaminants found in all aquatic ecosystems across the world. One of the methods to monitor levels of Hg contamination in an ecosystem is using sentinel organisms: abundant and widely distributed organisms within the food web that accumulate contaminants in body tissues without negative effects. Riparian spiders are a potential sentinel organism for the study of Hg contamination in aquatic ecosystems, as they accumulate mercury through the consumption of contaminated emergent aquatic insects. The present study will examine the effects of spider body size, sex, and season on 2 taxa of orb-weaving spiders [Family Araneidae: Larinioides sp., Metazygia sp.]. 575 orb-weaving spiders were collected from a boat dock on the South side of Eagle Mountain Lake, Texas, USA, from May to September 2019. The spiders were preserved in 95% ethanol and sorted based genera, month collected, sex, and size class. Size class was determined by measuring the spiders front left leg length (tibia + patella) and served as an indicator of body size. Mercury contamination will be analyzed through direct Hg analysis.

Plants with threatened habitats and fragmented populations may require repatriation efforts to maintain healthy populations. Populations of Sarracenia alata, the pale pitcher plant, are severely fragmented, and the species is near threatened. A complete understanding of its reproduction will be crucial in establishing and maintaining healthy populations. The goals of this study were to determine if 1) S. alata is capable of selfing (reproducing with pollen from the same individual); 2) S. alata is capable of autogamy (selfing without intervention); and 3) pollen load affects reproductive success. We used seed set to measure individual fitness. Thus, it was necessary to determine a reliable method of counting seeds. Two methods were examined, and these gave statistically similar results. We found that while S. alata is capable of selfing, it is not autogamous. Seed set was significantly higher in outcrossed individuals than in selfed individuals . In 2019, plants receiving supplemental pollen yielded more seeds than those in either the control group or a group in which pollinator access was restricted. During 2021 (a year with higher pollinator activity), there was no significant difference between the number of seeds produced by control plants and those receiving supplemental pollen. This study demonstrates the important role of pollinators in maintaining healthy populations in this system.

Migration, which is defined as the seasonal movement for survival or reproductive advantage such as access to resources, is a behavioral phenomenon exhibited by many species including the salmonid Oncorhynchus mykiss. More commonly known as rainbow trout, O. mykiss exists in two life histories: migrants (steelhead trout), and residents (rainbow trout). While there are many factors that contribute to this variation in migration behavior, one of the reasons is their genetic makeup since there is an apparent correlation between the migratory behavior of parents and their offspring. The primary objective of this research project is to identify single nucleotide polymorphisms (SNPs), or genetic differences, which are associated with migratory behavior in rainbow trout. To that end, I used whole genome sequence data from five migrant and five resident rainbow trout. These data were aligned to the trout genome and used to locate genetic differences between the two migratory types. Quantitative PCR (DMAS-qPCR) approaches were used to validate the SNPs and genotype them in a larger set of twenty-five migratory steelhead. Research findings exhibited that Sashin Lake is producing smolts (young migratory steelhead) that are successfully returning to the lake and reproducing at the end of their life cycle. Additionally, while there was not a significant difference seen in terms of marine survival between the sexes, females were more likely to migrate compared to their male counterparts due to the reproductive advantage and greater access to resources that migration offers. This data will support future studies observing trout migratory behavior with larger sample sizes and from different generations and settings and will benefit conservation studies regarding population decline in migratory species.

BRCA1 is a gene whose protein (also named BRCA1) is found throughout all human cells and engages in DNA repair, cell cycle regulation, gene transcription regulation, and apoptosis. However, mutations in BRCA1 typically confer a higher risk of cancer in estrogen-responsive tissues, including breast epithelial tissue. This increase in incidence of tissue-specific cancers is thought to be in part due to the role of BRCA1 in the estrogen response pathway and interaction with the estrogen receptor alpha (ERα). Previous studies identified possible regions of each protein involved in the binding interface between BRCA1 and ERα. Using these regions (amino acids 177-240 in BRCA1 and the ligand binding domain of ERα) as our constructs, our studies further analyzed the molecular details of this direct interaction and determined methods conducive to studying the BRCA1-ERα interaction. A pull down assay qualitatively confirmed binding between the constructs of BRCA1 and ERα. Data collected from NMR spectroscopy reaffirmed the direct interaction between BRCA1 and ERα first seen in the pull down assay and provided evidence demonstrating that the presence of estrogen in the samples increased binding affinity. Finally, fluorescence spectroscopy of quenching experiments confirmed the previous two results – that a direct interaction between the constructs of BRCA1 and ERα used occurs and the binding affinity increases in the presence of estrogen – and allowed us to describe the binding curve of the system being studied. The molecular details confirmed here provide further avenues of study, such as documenting variants of unknown significance or studying the role estrogen plays in the function of the BRCA1-ERα complex, which could lead to novel findings that expand our understanding of the role either protein plays in cancer development.

Alzheimer’s disease (AD) affects about 6 million Americans, and hallmark pathologies of AD include amyloid beta (Aβ), inflammation, and oxidative stress. Microglial cells (MGCs) are brain cells that function like immune cells, and they respond to Aβ by secreting pro-inflammatory cytokines. Cytokines induce inflammation at sites of infection, and Aβ continually increases inflammation, resulting in neuronal death. Inflammation is also connected to oxidative stress, and prior research has demonstrated that Nrf2 (a transcription factor) protects cells from oxidative stress by increasing antioxidant enzymes. We will test potential benefits of molecules with antioxidant capabilities, created by Dr. Green (TCU Chemistry), on inflammation and Nrf2 expression in MGCs. Previously, we demonstrated that these compounds, L2 and L4, are powerful antioxidants that protect MGCs from oxidative stress. Currently, we aim to study the effects of L2 and L4 on inflammation, Nrf2 expression and heme oxygenase-1 (antioxidant) production following an inflammatory insult. We will pre-treat MGCs with different concentrations of L2 and L4, and then stimulate MGCs with lipopolysaccharide (LPS), a bacterial mimetic. Subsequently, we will measure pro-inflammatory cytokines, Nrf2 expression and antioxidant response genes. Overall, it is crucial for researchers to investigate effective therapeutics that could relieve AD symptoms, such as antioxidant treatment.

Testing of chemicals that enter our waterways is necessary to keep marine environments healthy. The current method of toxicity testing is the larval growth and survival (LGS) test, which exposes larval fish to varying concentrations of an effluent or chemical. Given recent legislation that calls for improvements in the welfare of animals used in toxicity testing, there is a need to identify alternatives to the LGS test. In light of this, the objective of the current study was to determine whether toxicity tests featuring fish embryos or shrimp could be used in place of LGS tests.

To accomplish this, we compared the results of the standard LGS test using inland silverside larvae with the results from two alternative tests, a mysid (e.g., shrimp) test and an inland silverside fish embryo toxicity (FET) test. The results of this study show that both the mysid and FET tests are promising alternative testing methods to the LGS test. The adoption of either test type will meet legislative goals and improve the welfare of fish used in toxicity testing.

The Effect of Body Size on Mercury Concentration of Orb-Weaving Spiders (Araneidae) from the Clear Fork and West Fork of the Trinity River

Authors: Tyler Williams, Iris Schmeder, Morgan Capone, Matthew Chumchal, Andrew Todd, Ray Drenner, Cale Perry, Tori Martinez, Macyn Willingham, Robby Peterson, Chris Allender

Mercury (Hg) is a contaminant threatening all ecosystems. Inorganic Hg is released into the atmosphere from power plants and artisanal gold mines before being deposited over the landscape. Inorganic Hg deposited in the water can be converted by aquatic bacteria to methylmercury (MeHg). Methylmercury is one of the most toxic forms of Hg due to its capability of bioaccumulating within the tissues of organisms. Overexposure of methylmercury can cause damage to the nervous, genetic, and enzyme systems in the body, leading to a multitude of health complications. Evaluating the amount of Hg in an ecosystem, and thus the risk to organisms, is not straightforward. For example, the concentration of Hg in water or sediment may not be representative of aquatic organisms’ exposure to Hg because not all the Hg in water or sediment is bioavailable. As a result, scientists measure Hg concentrations in sentinels, defined as: an organism that can accumulate Hg within its tissue without significant adverse effects and serve as a representation of the level of Hg present within an ecosystem. Riparian spiders consume emerging aquatic insects and are therefore sentinels of Hg contamination in aquatic ecosystems. The objective of the study was to evaluate the concentration of total Hg in orb-weaving spiders (Family Araneidae) from the Clear Fork and West Fork of the Trinity River and determine how Hg concentration changes with spider body size. Spiders were preserved in 95% ethanol and body size was measured. Spiders were then dried and analyzed using a Direct Mercury Analyzer (DMA).

The long-term goal of exploring macrocycles is to be able to produce drugs that can interfere with certain protein-protein interactions within cells. This strategy could have the potential to change the way scientists think about drug design. Aspartic acid is a particularly useful to incorporate because it is one of the top five amino acids that contribute to binding at protein-protein interfaces. The acid sidechain of aspartic acid presents significant challenge because of the potential for side reactions. This research has established that an aspartic acid macrocycle can be synthesized quickly in three steps. The route is remarkably efficient and has the characteristics of those that could be used to make drugs. This poster details the chemical synthesis and characterization of this molecule, discusses potential side reactions, and identifies the next steps in advancing this project.

N-terminal acetylation plays an important role in the stability, activity, and targeting of proteins in eukaryotes. Most proteins expressed in bacteria are not acetylated, although the N-terminal acetylation is critical for the activities of a handful of biologically important proteins. Therefore, it is of practical significance to control N-terminal acetylation of recombinant proteins in bacteria. This study is aimed to alter the substrate specificity of RimJ, a protein N-terminal aminotransferase (NAT) that is known to acetylate a few recombinant proteins including the Z-domain in E. coli. The RimJ-mediated protein acetylation occurs at a higher rate when the substrate’s N-terminal amino acid is small. Because of this narrow substrate specificity, RimJ is not applicable for a broad range of recombinant proteins. Based on the AlphaFold-predicted structure of E. coli RimJ (AF-P0A948_F1), we predicted that five amino acids (Y106, M142, N144, Y170, and L171) may recognize substrate proteins in the active site. We created RimJ variants, in which one or two of these five amino acids are changed to alanine, a small neutral amino acid, so that the active site becomes larger to accommodate substrate proteins containing bigger N-terminal amino acid residues. Then, the substrate specificity of RimJ was investigated by co-expressing two Z-domain variants T2I and S3K, which were not acetylated by the wild-type RimJ. The expressed Z-domain variants were purified by immobilized metal affinity chromatography and subsequently analyzed by mass spectrometry, by which a 42-Da mass increment indicates the presence of an N-terminal acetyl group. The RimJ single mutants such as N144A, M142A, and Y106A showed little acetylation on both T2I and S3K Z-domain variants. In contrast, the RimJ double mutants, Y106A M142A, Y106A N144A, and Y170A L171A showed higher acetylation rates on the Z-domain T2I variants. Little acetylation was observed for the Z-domain S3K variant by any of these double mutants. We also created more RimJ variants in which three different amino acids located on the other side of the active site were changed to alanine. These variants will be used to co-express the Z-domain variants, whose N-terminal acetylation patterns will be analyzed by mass spectrometry.

In the lab, molecules used as drugs are made either in solution (wherein the reactive agents dissolve) or on solid supports referred to as 'beads' (wherein reactive agents are washed over beads and become attached only to be liberated later). The virtue of bead-based synthesis comes with the savings in time and energy normally required to purify the reaction products. That is, solution phase synthesis is work intensive. Here, a route to cyclic molecules synthesized on beads is described. The molecules produced by these bead-based methods have already been prepared in solution for comparison. In addition to evaluating the relative efficiencies of these two routes, the bead-based method can be used to rapidly make 100s-1000s of cyclic molecules. Such numbers are not possible using solution phase methods due to the burdens of purification. The effort relies on tethering an acetal to a reactive bead, followed by a protection and deprotection sequence, the addition of an amino acid using standard peptide coupling strategies and a reaction with a core group that offers the potential for the attachment of 100s-1000s of different groups. Cleavage of this linear molecule from the bead leads to spontaneous cyclization to the desired products. The products will be characterized by NMR spectroscopy and mass spectrometry as well as be assayed for biological activity in a disease model of breast cancer.