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.