Mycobacterium abscessus is a non-tuberculous mycobacterial (NTM) species that causes severe pulmonary infections, particularly in immunocompromised patients and those with preexisting lung diseases such as cystic fibrosis. Treating M. abscessus infections is challenging due to its intrinsic antibiotic tolerance and capacity to develop multidrug resistance. To identify novel molecules that can target this pathogen and enhance current treatments, we screened a library of FDA-approved drugs (n = 2,400). Our data shows that Netupitant, a drug commonly used to prevent chemotherapy-induced nausea and vomiting, exhibits potent antibacterial activity against a broad range of M. abscessus clinical isolates, including multidrug-resistant strains, with a minimum inhibitory concentration (MIC) ranging from 4 to 16 µg/mL. Furthermore, in combination with amikacin, a standard treatment for M. abscessus infections, Netupitant demonstrated strong synergistic interactions, as confirmed by checkerboard microdilution and time-kill assays. These findings highlight Netupitant’s potential as a novel therapeutic option for M. abscessus, particularly in combination with existing antibiotics. Future studies exploring its mechanism of action and in vivo efficacy could further advance antibacterial drug discovery for difficult-to-treat NTM infections.

This research study explores the evolutionary adaptations of the TLR7 protein receptor in primates in relation to Flavivirus (Yellow Fever) recognition. TLR7, an important receptor in the immune system, is essential for recognizing single stranded RNA viruses such as Flavivirus. Given the growing prevalence of Yellow Fever in tropical climates, I hope to understand how environmental factors shape immune response. I hypothesize that ecological niches, particularly wet vs dry climates, play a crucial role in the evolution of the TLR7 receptor among primate species. Since Yellow Fever is transmitted by mosquitoes, which thrive in wet climates, these environments are likely driving the transmission of the virus. As a result, wet climates may exert selective pressure on the evolution of TLR7 to enhance immune responses against Yellow Fever in regions where mosquitoes are prevalent. To conduct comparisons of the TLR7 receptors, I will be running Blast, a bioinformatics software that compares genomic sequences across different species. This tool will allow for identification of both conserved and divergent regions in the TLR7 sequences from primate species, including humans, that inhabit wet and dry climates. These variations could reveal evolutionary adaptations influenced by ecological pressures. Through these sequence comparisons, I aim to explore how differences in TLR7 might affect susceptibility to Yellow Fever and other similar viral infections. Understanding how ecological conditions shape immune receptor evolution could also improve our ability to predict how different populations might respond to emerging infectious diseases.

Plants' defense mechanism against herbivory is integral to both resistance in nature and the global food supply. Glycine max or soybean, is one of the most widely grown crops in the world, and suffers substantial losses from pests, including many Lepidopteran species. Related legumes, including cowpea, and common bean, can respond to Lepidopteran herbivory by detecting Inceptin-11 (In11), a short peptide found in larval oral secretions. The protein responsible for this ability, Inceptin Receptor (INR), is not found in soybean. The aims of this project are two fold, firstly introducing INR into soybean lines and testing for improved resistance to Lepidopteran herbivory, and secondly studying the effects of INR on defense gene expression in soybean, in order to better understand mechanisms of herbivory resistance. The first step in this project was to create soybean lines which consistently express INR. This was done by sending our INR construct to collaborators, who used it to inoculate multiple soybean lines, then breeding the corresponding lines until response to In11 was seen in all offspring. We will then test larval beet armyworms (Spodoptera exigua) on both INR- and INR+ lines. We expect the INR+ lines to have significantly lower S. exigua growth, indicating an improved immune response. We are also infiltrating INR- and INR+ lines with both In11 and flg22 (a well studied bacterial elicitor) for RNA sequencing of the early immune response. We expect genes involved specifically anti herbivory mechanisms being upregulated when compared to flg22. These two prongs allow us not just to demonstrate the viability of stable transgenic herbivory resistant lines, but to uncover the molecular mechanisms involved in that resistance, allowing for future scientists to better engineer the next wave of pest resistant crops.

Dopaminergic signaling within the striatum plays a crucial role in modulating reward and aversion, shaping behaviors such as food-seeking and consumption. While striatal dopamine release has been implicated in reinforcement learning and decision-making, the spatial and temporal dynamics of dopaminergic activity along the anterior-posterior axis of the striatum during consummatory behavior remain poorly understood. We investigated the role of dopamine in the striatum during the consumption of multiple solutions by employing a trial-based multi-spout behavioral paradigm with head fixed mice. To record the dopamine activity in the ventral and dorsal striatum, we utilized multi-site fiber photometry to record the fluorescent biosensor GRAB-DA2m along the anterior-posterior axis. Food restricted mice were given varying concentrations of sucrose as rewarding stimuli, while water restricted mice were given varying concentrations of sodium chloride as aversive stimuli. Our results revealed that dopamine responses scaled more across concentrations in the anterior regions of the striatum compared to the posterior regions. Additionally, we found more distinction between dopamine responses for the various concentrations of the aversive solution compared to the rewarding solution. Lastly, posterior striatal dopamine responses had a more rapid onset upon stimulus consumption, whereas anterior regions exhibited delayed responses, highlighting region-specific temporal differences in dopaminergic encoding. These findings refine our understanding of dopaminergic circuitry within the striatum and how dopamine-mediated responses to rewarding and aversive stimuli regulate feeding behaviors. By exploring this pathway, we offer potential insights into the mechanisms underlying disorders characterized by dysregulated reward including eating disorders and obesity.    

Preterm birth is a leading cause of neonatal morbidity and mortality, with intra-amniotic infection and inflammation being major contributors to early preterm labor (PTL). Despite ongoing research aimed at reducing inflammation in neonates, most studies have focused on post-delivery while few have been done prior to delivery. IL-1 is a central upstream mediator of inflammation in the amniotic cavity and the neonate. IL-1 is a key cytokine that is responsible for induction or propagation of the cytokine cascade responsible for PTL. Rytvela, an interleukin-1 receptor antagonist made up of seven D-amino acids, acts as a selective antagonist of IL-1 signaling, which could be used to act as a therapeutic approach to reduce inflammation and prevent PTL. The purpose of this experiment is to determine if interleukin-1 (IL-1) is a key molecular target for the development of antenatal therapeutics to prevent PTL and fetal injury. We hypothesize that Rytvela administered intravenously to the mother will cross the placenta and be detectable in the amniotic fluid and fetal plasma, suggesting that Rytvela could effectively block IL-1 signaling in the fetus and therefore reduce fetal inflammation. Maternal blood plasma samples were drawn at Day 1, 2, 6 and 10 post infusion. To confirm the transfer of Rytvela to the fetus, we used liquid chromatography-mass spectrometry (LC-MS) to detect the drug, looking at integration, peak identification, and backlog pressures to see if Rytvela is detectable in maternal plasma. Rytvela was detected and luminex plates were run to measure cytokine levels. After GBS infection, Il-1 beta and Il-23 concentrations increased. After Rytvela administration, the concentration of the pro-inflammatory cytokines decreased. Future directions will involve measuring cytokine levels at these time points and correlating them with Rytvela infusion to evaluate the drug’s impact on maternal-fetal inflammation

Plants defend themselves against invading pathogens and herbivores using immune receptors that detect molecular signals associated with danger. Two types of plant immune receptors are the leucine-rich repeat (LRR) receptor, receptor-like kinases (RLKs), and receptor-like proteins (RLPs) through which signal transduction may proceed through the cell. Coreceptors like SERK3/BAK1 and SOBIR1 pair with LRRs to facilitate intercellular communication. We studied a specific LRR receptor, the inceptin receptor (INR) that recognizes an eleven amino acid-long peptide chain known as inceptin-11 (in11). Given the close interaction of LRR and its coreceptors, and considering that we still don’t fully understand how INR recognizes in11, we investigated its three-dimensional structure to analyze the mechanism of ligand binding and signal activation. Through predictive modeling in Alphafold of homolog RXEG1, a carboxy-terminal loop out domain was identified as a facilitator in the binding interaction between INR and the BAK1 coreceptor. To explore this mechanism, we introduced targeted mutations in the domain’s K-X5-Y motif to potentially change its ability to recruit BAK1. When a ligand attaches to a receptor, its conformation change allows signals to pass through the cell membrane. We constructed a library of 36 single and double mutants in the K-X5-Y motif and coexpressed them with a luminescence-based reporter construct in Nicotiana benthamiana to screen their activity. We expect that a mutation at K or Y or both will affect BAK1 recruitment, leading to phenotypes that are hypersensitive or inactive. Insight into LRR-RLP coreceptor interactions could open doors to further INR-immunology research alongside better modeling of BAK1 protein binding. Targeting immune-related peptides in this screen could significantly advance cultivation programs for INR expressing organisms.

Pregnant women infected with influenza A virus (IAV) are at higher risk of morbidity, mortality, and poor fetal outcomes. However, the difference in the pathogenesis of IAV between pregnant women and non-pregnant women remains inadequately understood, primarily due to the lack of animal studies that use a translational model of infection. I hypothesized that higher IAV viral load and Type I interferon concentrations would be observed in the lungs and bronchoalveolar lavage of pregnant pig-tail macaques compared to non-pregnant macaques, and that correlating these metrics would yield different results across groups. We inoculated pregnant (n=11) and non-pregnant female (n=18) pig-tail macaques (Macaca nemestrina) with IAV H1N1 (A/California/07/2009) and euthanized them at 5 days post-inoculation, when we expected to observe peak lung pathology. We tested pulmonary function at baseline and study endpoint and conducted clinical assessments daily. I extracted RNA and performed quantitative polymerase chain reactions on the samples to calculate viral load. I also performed enzyme-linked immunosorbent assays to quantify concentrations of Type I interferons (IFN-α, IFN-β). Lastly, I analyzed pulmonary physiology data and clinical assessment scores as a reliable measure of disease severity. A bi-modal distribution of viral load was observed in the lungs of pregnant animals (high>9e5 copies/mg; low<2e4 copies/mg), which was not observed in non-pregnant animals.  When correlating viral load at 5 days post-inoculation with Type I IFN in the lung of the pregnant animals, I found a significant positive correlation between IFN-β and viral load in both the lungs (ρ=0.8, p=0.03) and BAL (ρ=0.9, p=0.02). These results suggest that despite a strong IFN-β response in the lung, a high viral load persisted in the pregnant animals. Next steps could explore whether the kinetics of the pulmonary innate immune response is delayed in pregnancy, which impairs viral clearance. 

Chloroplasts are central to plant immunity and act as a hub for immune signalling and defence-related hormone synthesis. The essential chloroplast-localized protein FtsHi1 is a component of the FtsHi import motor and is vital to translocating proteins across the chloroplastic membrane. Viral-induced gene silencing (VIGS) of FtsHi1 in Nicotiana benthamiana results in a bleached phenotype, indicative of decreased translocation of essential chloroplastic proteins and decreased chlorophyll synthesis. Previous work identified herbivore-induced kinase 1 (HIK1) as a potential interactor of FtsHi1. HIK1 is a receptor-like cytoplasmic kinase (RLCK) implicated in the immune response to caterpillars. FtsHi1 contains a predicted RLCK phosphorylation site, indicating possible phosphorylation of FtsHi1 by HIK1, which could promote defence signalling over photosynthesis. This project aims to test the impact of phosphorylation on FtsHi1 function and its role in protein translocation across the chloroplastic membrane. This work utilises engineered FtsHi1 variants, which mutate the serine phosphosite to either mimic FtsHi1 phosphorylation (Ser→Asp), prevent it (Ser→Ala), or recapitulate the wild-type protein sequence. To test the phosphosite mutation effects, FtsHi1 VIGS knockdown of N. benthamiana leaves will be transiently modified using Agrobacterium tumefaciens bacteria to express WT or phosphorylation-modified FtsHi1 constructs. The resulting colour phenotype for each construct will then be compared to the bleached phenotype of TRV2:FtsHi1 plants and the phenotype of wild-type plants. I hypothesise that the FtsHi1 phospho-null mutant will result in a rescue phenotype similar to wild-type FtsHi1, whereas FtsHi1(phospho-mimic) will retain the bleached phenotype.Examining FtsHi1 phosphorylation enhances our understanding of its potential interaction with HIK1 in herbivory-induced plant defences. Future studies will explore FtsHi1's role in defence mechanisms, with implications for engineering herbivory-resistant crops. 

Plants utilize molecular patterns in order to detect threats to the plant. Through the recognition of molecular patterns by their associated receptor, plants are able to initiate an appropriate immune response, measurable by the increased production of reactive oxygen species (ROS). In the model organism Arabidopsis thaliana, the pathogen associated molecular pattern flg22 is detected by the pattern recognition receptor Flagellin Sensitive 2 (FLS2) to initiate intracellular signaling. The immune signaling kinase Botrytis-Induced Kinase 1 (BIK1) is then phosphorylated by FLS2 to transduce the immune signal, initiating ROS production. However, A. thaliana lacks a group of immune signaling kinases related to BIK1 called Herbivory-Induced Kinase Like Kinases (HLKs), which are present in multiple species including tobacco (Nicotiana benthamiana) and common bean (Phaseolus vulgaris). The goal of this project is to determine the role of HLKs in immune signaling. To investigate the role of HLKs in immune signaling, A. thaliana were transformed with transgenes to express HLKs or overexpress BIK1. Stably transformed A. thaliana lines were then bred to produce progeny that are homozygous for the transgenes. These lines are treated with flg22 in order to initiate an immune response. ROS is used to measure the immune response of each transgenic line, where HLK expressing individuals are compared with BIK1 overexpressing individuals as a control group. I anticipate that HLKs will increase the ROS production when compared with the controls, signifying an increased immune response, since HLKs are related to the native BIK1. An understanding of the role of HLKs in FLS2 immune signaling in the model organism A. thaliana can be applied to crop species that employ HLK mediated immune signaling.

According to the Centers for Disease Control and Prevention, the U.S. has more than 2.8 million antibiotic-resistant infections each year. The rise of multidrug resistance in bacteria poses an urgent clinical threat contributing to these various infections. UPAB1 is a specific strain of a notoriously drug-resistant bacteria Acinetobacter baumannii associated with catheter-associated urinary tract infections (CAUTI). UPAB1 infects the urinary tract through the introduction of a foreign object, such as a catheter. In response, the immune system coats the catheter with fibrinogen, a glycoprotein complex that assists in wound healing. UPAB1 uses its bacterial adhesin proteins, such as Abp2D, to bind to fibrinogen, deplete essential nutrients, and infect the urinary tract. By designing Abp2D inhibitors as de novo miniproteins, we hypothesize that A. baumannii will be prevented from establishing a bacterial infection and allow us to offer a potential alternative in combating antibiotic resistance in CAUTIs. Targeting UPAB1 Abp2D, we first developed designs of Abp2D inhibitors utilizing computational software like RoseTTAFold Diffusion (RFdiffusion) for miniprotein backbone design, ProteinMPNN for sequence design, and AlphaFold2 (AF2) for structure prediction of the sequences to validate and filter. Afterwards, in the laboratory, we expressed and purified the miniprotein designs. We are currently testing these designs as Abp2D inhibitors via E. coli cultures to determine their success in binding to UPAB1 Abp2D.