ClpX is a regulatory ATPase that functions along with ClpP as part of the intracellular bacterial ClpXP protease. Previous research from our group has shown that genetic loss of ClpX (∆ClpX) in Bacillus anthracis Sterne increases susceptibility to antimicrobial agents that target or interact with the cell wall including penicillin, daptomycin, and LL-37. In order to gain a better understanding of ClpX function in B. anthracis Sterne, a microarray analysis comparing WT and ∆ClpX gene expression was performed in B. anthracis. We found that LrgAB, a negative regulator of autolysis, was significantly downregulated in the ∆ClpX mutant and this finding was confirmed with QPCR. In order to determine whether LrgAB also had a role in antibiotic resistance in B. anthracis, we made a genetic deletion of LrgAB (∆LrgAB) and found it has similar phenotypes to ∆ClpX in B. anthracis. To see if these findings were consistent in other gram- positive pathogens, we expanded our research to Staphylococcus aureus, the leading cause of skin and soft tissue infections. We constructed a ∆ClpX mutant in the Newman strain of S. aureus and found it also exhibited sensitivity to cell wall active antimicrobial agents. Loss of ClpX in S. aureus also resulted in decreased expression of LrgAB by QPCR. Lastly, we examined a S. aureus ∆LrgAB mutant and observed an increase in antibiotic susceptibility. We conclude that ClpX plays a role in resistance to cell wall active antimicrobials in both B. anthracis and S. aureus, and that this is connected to its regulation of LrgAB.

Breast cancer is a growing problem in the United States and worldwide. It takes the lives of approximately 40,000 U.S. women a year. 1 in 8 U.S. women will develop breast cancer during the course of their lifetime and it continues to be the most commonly diagnosed cancer in women. Clearly, this is a serious issue that must be solved. Current chemotherapy treatments often result in widespread cell death, including the killing of healthy cells. Therefore, it is necessary to find alternative treatments that specifically target cancer cells. Many breast cancer cells over express estrogen receptors, which are vital to the rapid cell division and growth of tumors. Estrogen is a steroid hormone that enters the cell, binds to its receptor, translocates to the nucleus, and leads to gene expression. Previous work from our group has resulted in the development of a drug which targets estrogen receptor positive breast cancer cells called Est-3-Melex. The drug contains a DNA methylating group (Melex) conjugated to estrogen. The mechanism of action of the drug is by the binding of the estrogen portion of the molecule to its receptor that ultimately translocates to the nucleus. While in the nucleus, the Melex portion of the compound is brought in close proximity to the DNA and methylates the adenines, eventually resulting in cell death. Essentially, this is a receptor targeted cancer therapy. In order to test the toxicity of this drug, we utilized a MTT cytotoxicity assay, which quantifies the amount of cell death. Est-3-Melex was more toxic to cancer cells that overexpressed the estrogen receptor compared to those that did not. Treating the estrogen receptor positive breast cancer cells with excess amounts of estrogen inhibited Est-3-Melex-induced cell death. Fluorescence imaging was also utilized to visualize localization of the drug. A fluorescent tag was attached to Est-3-Melex and introduced into estrogen receptor positive breast cancer cells. The results showed the drug localized to the nucleus and this localization was inhibited by estrogen. Our results suggest that Est-3-Melex is effective in specifically killing estrogen receptor positive breast cancer cells by binding to the estrogen receptor. Additional investigations are underway to identify the mechanism of cell death.

The effects of the thyroid axis on metabolism, growth, and development are well documented. However, there is a paucity of information on the role of thyroid hormones in the development of the immune system. Therefore, the goal of this study was to determine the effects of early life stage exposures to thyroid-altering chemicals on the developing immune system using the fathead minnow (Pimephales promelas) as the model, an organism commonly used in toxicity testing. This was accomplished by measuring differential expression of several immune-related genes in fish exposed to various doses of propylthiouracil (PTU, a thyroid-inhibitor) and thyroxine (T4, a thyroid-stimulator) sampled at 7 and 35 days post hatch (dph). Fish exposed to PTU exhibited significant increases in rag2 expression at 7 dph, decreases IgLC1 expression of at both 7 dph and 35 dph, and decreases in IgLC3 expression at 7 dph. In contrast, T4-exposed fish showed elevated rag1 and rag2 expression at both 7 and 35 dph, increased IgLC2 expression at 7 dph, and upregulation of ikaros at 35 dph. The results of this study indicate that exposure to thyroid altering chemicals influences the expression of several genes associated with proper immune system development, indicating that thyroid hormones regulate various aspects of immune development. These findings provide evidence that exposures to environmentally-relevant compounds that modulate thyroid function may lead to improper immune system development, which is likely to adversely affect overall organism health.

Recent research has identified dung beetles as bioindicator species found in a wide range of environments. Bioindicators function as monitors for the health of an ecosystem, which can be determined by analyzing the function, population, or status of the species in said environment. The purpose of our project was to determine if dung beetle diversity and abundance differed between primary and secondary rainforests. We conducted a study in the transition zone between tropical wet forest and premontane rainforest at the El Jamaical Field Station in Costa Rica. We acquired feces from both cows and horses near the field station. For trial 1, we made four bait traps using cow feces and one control for each of the forest types. Within each forest type, we placed the bait traps 25 meters apart. We then repeated the experiment using horse feces for trial 2. Traps sat for a period of 24 hours to allow dung beetles time to burrow into the traps. We then collected and processed the samples. Processing consisted of sifting and breaking down the feces in a meticulous manner to find, collect and identify all dung beetles present. We identified a total of 303 beetles in trial one and 0 in trial 2.

Mercury (Hg) is a hazardous contaminant that can be transferred from aquatic to terrestrial environments by emerging aquatic insects. Terrestrial predators, such as spiders, that live along shorelines of water bodies may consume emerging aquatic insects and become contaminated with Hg. Mercury-contaminated spiders may pose a risk to arachnivorous songbirds. The degree to which most families of spiders are contaminated with Hg and the risk they pose to songbirds is not well understood. The objectives of this study were to determine 1) Hg concentrations in two families of shoreline spiders (long-jawed orbweavers, [Tetragnathidae] and crab spiders [Thomisidae]) and 2) determine the risk these spiders pose to arachnivorous birds. We collected representatives from two families of spiders from the shorelines of 10 ponds located at the LBJ National Grassland in north Texas, USA. Both spider taxa in the present study were contaminated with Hg, however long-jawed orb weavers had significantly higher concentrations of Hg in their tissues than crab spiders (p < 0.001; average Hg concentration = 346 ng/g and 35.7 ng/g respectively). We calculated wildlife values for various songbirds to determine health risks that these Hg-contaminated spiders may pose to songbirds. Spider-based wildlife values revealed that one of the families of shoreline spiders, Tetragnathidae, had concentrations of MeHg high enough that they may pose a risk to arachnivorous songbirds that consume spiders along the shorelines of ponds.