C. difficile is a bacterium that releases two cytotoxins, toxin A and toxin B, which can cause cellular inflammation and apoptosis. C. difficile infections (CDI) are becoming more common and are often acquired during hospital visits, especially after antibiotic treatment. The most promising treatment currently is fecal microbiota transplants, but this treatment has had a couple cases of deadly side effects and its administration can be invasive. Based on a previous study done on the protective effects of various metabolites against CDI by the Koon laboratory, we further investigated whether the metabolite citrulline has potential as a treatment against CDI using mice models, human primary epithelial cells, and human primary macrophages. Our findings suggest that citrulline has an anti-inflammatory effect on CDI intestinal tissue and that this effect may be caused by citrulline inducing a decrease in MIP-1α secretion and an increase in IL-10 secretion. We will be expanding our study further by verifying these previous finding with more experimental rounds, testing on hamster models, and comparing the microbiome of treated and non-treated animal groups.

The first innate behavior to be expressed by embryonic C. elegans is a dorsal-ventral head oscillation that is governed by a group of six glutamatergic IL1 neurons. These neurons are mechanosensitive and synapse with head muscle cells, but their precise function in regulating this behavior isn’t known. To determine whether the neurons are muscle-excitatory or inhibitory, Genetically Encoded Calcium Indicators, which fluoresce in the presence of and by binding to calcium, can be used to compare intensity levels and see if they correspond to behavior. One such group of these indicators, GCaMP, offer different versions of the protein, with varying calcium affinity and signal intensity. However, a balance must be achieved with these indicators: calcium is important to the early development of the worms, so a GCaMP binding calcium too strongly can be lethal, but a GCaMP binding calcium too weakly might not give a detectable signal. Currently, GCaMP6f is used to image C. elegans embryos, but more recent versions of GCaMP have come out, namely GCaMP7s and GCaMP8s, reporting greater intensities and sensitivity. To determine the viability of their use in C. elegans, we injected them on an unc-120 vector, to observe expression on muscle cells. To date, we have established two strains of worms with GCaMP7s and are looking to compare them to our strain with GCaMP6f. A strain carrying GCaMP8s has been generated, but fluorescence signal was not observed, we are now working to determine whether this is due to expression or functional issues with the construct.

Carbon dioxide (CO2) influences the behavior of Caenorhabditis elegans (C. elegans) in a context-dependent manner. Well-fed C. elegans show an aversion to CO2, while starved animals or well-fed animals maintained at high CO2 show attraction to CO2. The molecular mechanisms governing the shift from CO2 aversion to CO2 attraction are poorly understood. EGL-4 is a cGMP-dependent protein kinase that has been shown to affect C. elegans behavior. The egl-4 mutant strains egl-4(n478) and egl-4(n479) harbor a missense and nonsense mutation, respectively. Based on its role as a regulator of valence in odor-evoked systems, we hypothesized that egl-4 plays a role in mediating CO2-evoked valence. The CO2 chemotaxis assay was used to the CO2-evoked behavioral response. C. elegans are placed onto a plate, and CO2 and air are pumped into opposite sides. Animals in scoring regions are counted to analyze behavior. Well-fed egl-4 mutants maintained at ambient CO2 levels were repulsed by CO2, consistent with wild-type animals. Starved egl-4(n479) animals maintained at ambient CO2 were attracted to CO2, a result consistent with wild-type animals. However, the egl-4(n478) animals showed no preference. Well-fed egl-4 mutants maintained at high CO2 levels were repulsed from CO2 while wild-type animals were attracted to CO2. Taken together, these data support the conclusion that egl-4 plays a role in the CO2-evoked valence switching in C. elegans. Going forward, cell-specific rescue experiments, genetic ablation analysis, and calcium imaging will be used to identify and functionally characterize the neurons in which egl-4 is acting to regulate CO2 response valence.

Primary Hyperoxaluria Type 1 (PH1) is a lethal, autosomal recessive disorder that occurs when mutations occur in the alanine-glyoxylate aminotransferase (AGT) enzyme. A subset of mutations cause a functional version of the protein to mislocalize from peroxisomes to mitochondria. Mislocalization to mitochondria prevents conversion of glyoxylate to glycine in liver cells. The mutation leads to kidney failure when glyoxylate moves into the cytosol and is converted to oxalate. Our project seeks to identify methods through which small molecules restore AGT function. We hypothesize that an existing compound, MitoBloCK-11 (mitochondrial import blocking peptides from the Carla Koehler laboratory), restores the trafficking of mutant AGT to peroxisomes by blocking import into mitochondria in a human cell model. To test this, we have developed a screening system in liver HepG2 cell lines. A split-GFP system has been used in which AGT contains the terminal GFP beta-sheet (AGT-GFP11). The majority of GFP (GFP1-10) is co-expressed and targeted to the peroxisome. Small molecules that block AGT-GFP11 import into mitochondria and restore peroxisomal import result in the reconstitution of GFP and subsequently fluoresce in the peroxisome. Treatment with 1 uM MitoBloCK-11 and detection by fluorescent microscopy indicated that peroxisomes had robust green fluorescence, indicating the AGT was redirected from mitochondria to peroxisomes. Future studies will use a molecule screening approach to identify other compounds that may be more suited for drug development. We propose to develop a high content screen using microscopy to confirm reconstitution in the peroxisome and restore AGT localization.

Microglia are immune-like central nervous system cells(CNS) that have been shown to undergo changes with age. Hallmark features of brain aging include regional changes in brain volume and composition, as well as altered neuronal network activity patterns. Microglia have recently been shown to regulate the extracellular matrix, a key structural support system in the brain. Although there has been evidence that microglia interact with the ECM, there is little data regarding the impacts of age on microglia-ECM interactions and how these changes might impact memory. Our goal is to map regional activation patterns in aging mice and assess microglial-ECM interactions within these regions. To accomplish this, we developed a reward-based spatial memory task. Following behavioral testing, we collected the brains through perfusion and sliced them using a vibratome. Using immunohistochemical staining techniques, a total of 8 brain sections per animal from anterior to posterior were stained for the immediate-early gene cFos in order identify regional activation patterns associated with the spatial memory task. Sections were imaged using a Leica scanning microscope and analysis was performed in Fiji image analysis software. Our results indicate that the most activated regions were Anterior Piriform, Cornu Ammonis, and Anterior Cingulate cortex. While the least activated were Nucleus accumbens, Dentate Gyrus, and posterior Piriform regions. It is important to note these regions contain varying ECM compositions. Together These data will focus future proteomic and image-based experiments on behaviorally relevant brain regions, facilitating a thorough investigation of the impacts of age on microglial-ECM/neuronal function.