Benzalkonium Chlorides (BACs) are widely used antimicrobial disinfectants in a variety of settings, including large scale food processing and consumer environments. Persistent usage of BACs raises concerns about the potential disruption of the gastrointestinal microbiota, an increasingly recognized regulator of an individual’s health. Furthermore, the gut microbiota has been shown to regulate drug metabolizing enzymes (DMEs) and the Gut-Liver Axis is a known prominent crosstalk pathway. Previous work in our lab has found BACs are capable of altering gut microbiome composition in BAC exposed C57BL/6 male and female mice, with notable differences between the male and female sexes. Therefore, we hypothesize that exposure to the BACs can alter the composition of gut microbiota, leading to sex specific changes in bile acid homeostasis as well as the metabolic phenotype and DME expression of the liver. In this study, we exposed male and female mice to C12- and C16-BACs at 120 ug/g/day for one week via oral dosing. Additionally, through a targeted bile acid quantitation analysis, we found sex specific decreases in secondary bile acids in BAC-treated mice. This finding is supported by decreases in bacteria known to metabolize primary bile acids into secondary bile acids, such as the families of Ruminococcaceae and Lachnospiraceae. We also aim to elucidate both transcriptomic (RNA sequencing) and functional (enzyme activity assays) analyses of the harvested livers from both male and female cohorts. Upstream pathway analysis from the results of these analyses is expected to yield sex specific differences in the downregulation of genes responsible for a variety of pathways such as protein digestion and absorption and transcriptional regulation in cancer. This study is expected to provide novel insights into the sex specific alterations in the relationship between the gut microbiome and liver caused by BAC exposure and the mechanisms underlying BAC toxicity.

Cell death and the processes surrounding it are essential parts of life. Ferroptosis is a distinct type of regulated cell death characterized by increased lipid peroxidation leading to cell membrane damage. The exact mechanism of ferroptotic death is currently unknown, so research is underway to discover pathways that can modulate ferroptosis. The goal of this project is to determine how different isoforms of the ACSL gene impact ferroptosis mediated by non-conjugated and conjugated polyunsaturated fatty acids (PUFAs). ACSL4 (long-chain acyl-CoA synthetase 4) is a gene of importance in ferroptosis because it incorporates PUFAs into the cell membrane. The membrane-incorporated PUFAs can be oxidized via lipid peroxidation in the cell, leading to membrane damage and eventual cell death. It has been found that knocking out or silencing the ACSL4 gene can make cells resistant to ferroptosis. I compare how ACSL4-knockout cells react to different PUFAs, and how these PUFAs sensitize wild-type and ACSL4-knockout cells differently when treated with ferroptosis-inducing drugs. Cell viability assays are a way to measure the amount of cell death in response to a treatment. I perform these assays to create dose-response curves for different lipid and drug treatments and use flow cytometry to quantify the amount of lipid peroxidation. These assays help establish a baseline comparing the response of wild-type and ACSL4-knockout cells to different PUFAs and ferroptotic drugs. Preliminary results demonstrate greater percent viability in two different knockout cell lines compared to wild-type cells when treated with the drug RSL3 and arachidonic acid. ACSL4-knockout cells are expected to have decreased cell death if they are protected from ferroptosis. Results will demonstrate the extent to which knocking out the ACSL4 gene affects cell survival. Ultimately, ferroptosis is a process of interest due to its therapeutic potential in treating tissue damage and as a targeted cancer therapy.

Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza virus. HA binds to sialic acid receptors on host cells and mediates membrane fusion, allowing the virus to enter the cell. This project investigates how mutations associated with species crossover affect viral fusion mechanisms in influenza HA. HA from the past outbreaks of H5N1 influenza strains in Vietnam in 2004 (VN04) and Indonesia in 2005 (IN05), are being compared to an HA from an on-going avian influenza outbreak using a strain isolated in Colorado in 2022 (CO22). Through site-directed mutagenesis, mutations that affect acid stability and affinity for the human receptor were added to HA based on the VN04 and IN05 strains; we are studying these effects in recombinant protein rather than on infectious virus. These mutations are believed to enable the virus to increase transmissibility among mammals including humans. To compare how HA from these H5 isolates with and without the adaptive mutations behave, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is being used to measure changes in deuterium incorporation on the protein backbone for specific peptide segments, giving a profile of local dynamics and structure throughout the HAs. By comparing the dynamic profiles for each as pH is lowered, mimicking acid-activation in host cell endosomes, we can probe how their structure in important fusion and human receptor-binding regions change as the fusion protein becomes activated. By comparing the structural dynamic changes of the WT and mammal-adapted, mutated IN05 and VN04 HA to the new CO22, we will be able to increase our understanding of the effect of the mutations that are associated with species crossover and hopefully be able to gain insight into the potential of this new avian influenza strain’s ability to become transmissible among humans.

Aging is associated with shifts in the composition of brain extracellular matrix chondroitin sulfate glycosaminoglycans (CS-GAGs). CS-GAGs are comprised of repeating glucosamine and N-acetylgalactosamine units that are either non-sulfated (0S-CS), mono-sulfated (4S-CS, 6S-CS), or di-sulfated (2S6S-CS, 4S6S-CS, 2S4S-CS/Dermatan) and participate in the regulation of brain plasticity. The mono-sulfated 6S-CS isomer is predicted to play a key role in the induction of circuit plasticity during neurodevelopment. Therefore, we asked whether this isomer also shows consistent age-related changes between wild-type mice and humans in the regions of the hippocampus and cortex. Our preliminary data generated from cohorts of mice ranging in age from 7 days to 2 years (50%M/50% F) reveal that 6S-CS abundance is highest at 7 days of age and declines with increasing age (9-22 mice/group). We analyzed the relative abundance of the 6S-CS isomer in n=57 hippocampal and cortical human tissue samples (age: newborn - 95 years, sex: 50%M/50% F). Initially, the human samples exhibited the highest abundance of 6S-CS isomer following birth (<1 month age) that then declined at >1M to 29 years of age, phenocopying the results from mice. However, in contrast to mice in which 6S-CS abundance decreased progressively with aging, we found that in humans, 6S-CS abundance began to increase starting at 30 to 99 years of age (R² = 0.84, p-0.0001). The biphasic model of changes in 6S-CS abundance in humans throughout normal aging was previously unknown. Collectively, these findings demonstrate that age-associated changes in brain extracellular matrix 6S-CS isomer abundance in human tissue do not reflect the age-related decline of 6S-CS isomers that occur in mice. Therefore, additional research is needed to establish the utility and robustness of using rodent models to study aging and other age-related extracellular matrix diseases in humans.

Half a million people develop drug resistant tuberculosis (TB) each year. Cases of drug resistant TB often result in poorer outcomes both healthwise and economically for patients, and many populations lack access to the resources needed to treat resistant TB. Increased antibiotic resistance has resulted in an urgent need to develop new, cost-effective drugs that are effective against Mycobacterium tuberculosis, the bacteria responsible for TB. In my research, I am using deep learning methods to design peptides that bind to the enzyme ClpP, a vital protease and known antibiotic target in M. tuberculosis. A class of drugs called Acyldepsipeptides (ADEP) have been shown to bind to ClpP and cause cell death in M. tuberculosis by preventing the formation of the ClpP complex with necessary ATPases, resulting in significantly lower proteolytic activity. We used the structure of ADEP as a basis for the peptide design and employed Rosetta, a macromolecular prediction and design software, to generate cyclic peptides bound to ClpP. I then used a sequence based deep learning tool to generate multiple sequences for each backbone design and computationally validated the resulting structures with AlphaFold, a highly accurate, machine learning based structure prediction tool. The structure of the ClpP binding interface resulted in it being a difficult target to design for with current deep learning methods. One peptide binder was predicted to bind to ClpP in our preliminary design rounds. We will chemically synthesize this binder and test it against ClpP in an enzyme inhibition assay. If the binder inhibits ClpP, it can serve as a basis for an effective and low cost drug that targets the ClpP enzyme in drug resistant TB. We will also expand and refine our design pipeline to produce more binder designs that can serve as viable drug candidates.

While vaccines help prevent infection from SARS-CoV-2, therapeutic drugs remain necessary to treat people who are infected. In my research, I develop peptide-based therapeutics, which are safe and readily bioavailable due to their low immunogenic response and low production cost. I help design peptide inhibitors against the Mpro enzyme, the main protease present in SARS-CoV-2. Mpro normally cleaves the virus’ pp1a and pp1ab viral polyproteins, which are important for viral replication and transcription. By creating a competitive inhibitor that binds to Mpro, I can prevent it from activating these proteins and thereby prevent viral proliferation. We began with computational design of peptide inhibitors using the stub-extension approach on the Rosetta Macromolecular Modeling suite, a peptide design computational platform. An effective drug both finds the active site of Mpro and binds to it more strongly than the natural substrate. First, our group started with selecting the most critical residues (stubs) that form well-characterized interactions in the active site. We then used Rosetta to add amino acids and cyclize the peptides. After generating cyclic backbones, we designed amino acid side chains with high shape and chemical complementarity to the active site. After filtering, we proceeded with the 50 best 7- to 11-mer peptide candidates. Afterwards, I used solid phase peptide synthesis to chemically synthesize these peptide designs, followed by cyclization and purification with High-Performance Liquid Chromatography. I helped to test their effectiveness in mass spectrometry-based inhibition assays. Overall, we have identified four promising peptides, and our most promising peptide is gzm1_1, which has an IC50 value (the concentration of peptide required to inhibit 50 percent of Mpro) of 0.076 µM. Through refining the structure of these inhibitors even more, I can improve their inhibitory effectiveness, enabling them to serve as the basis of an effective medication for people infected with SARS-CoV-2.