The Xpert Xpress SARS-CoV-2 assay is a rapid RT-PCR test developed by Cepheid during the early stages of the COVID-19 pandemic. However, with the emergence of novel variants, concerns have been raised about possible diminished sensitivity of RT-PCR assays that were developed using earlier strains of SARS-CoV-2. We sought to address these concerns by testing known positive Alpha, Beta, Delta, Gamma, and Omicron SARS-CoV-2 samples which, at the time of testing, were classified by the CDC as variants of concern (VOCs). Since these samples were stored in TE buffer, a medium not formally evaluated for use in the Cepheid Xpert Xpress SARS-CoV-2 assay, we also tested positive controls using both TE buffer and the standard viral transport medium (VTM). All samples were run using the Xpert Xpress SARS-CoV-2 assay per Cepheid's provided instructions. Testing of known VOC positives revealed no substantial reduction of PCR sensitivity when compared to a clinically certified RT-qPCR assay. Comparison of TE and VTM samples also revealed no reduction in performance when using TE buffer, validating the use of TE buffer to store SARS-CoV-2 samples. Our findings show that the mutations present in the current circulating VOCs do not lead to false negative patient results, indicating the Xpert Xpress assay may still be used for diagnostic testing.

Syphilis is a sexually transmitted infection (STI) caused by the bacterial spirochete Treponema pallidum subsp. pallidum. Syphilis is a multistage infection with the stages aptly named primary, secondary and tertiary syphilis and can also manifest itself latently and congenitally (LaFond & Lukehart, 2006). The exact immunology of syphilis has not been clearly defined. The purpose of this research was to elucidate antigens and immunogenic epitopes of interest to help characterize the immune response against T. pallidum. In this experiment, peripheral blood mononuclear cells were enriched and T. pallidum specific CD4+ T cells were isolated and expanded from a convalescent patient blood sample. I created recombinant proteins from select orfeomes Tp870 and Tp684 of the T. pallidum genome, which had previously been predicted to stimulated CD4+ T cells. Synthetic peptides comprising these two proteins were purchased and assayed with an ELISA. By measuring the reactivity of the CD4+ T cell reactivity to each, I was able to preliminarily characterize T. pallidum epitopes. This workflow is useful for epitope discovery and progressing the understanding of syphilis immunology.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder that affects people who have mutations in the PKD1 or PKD2 genes. The prominent feature of this disease is the formation of cysts in the kidneys. Despite ADPKD being genetically heterozygous, cystic tissue from patients with ADPKD also contains cells with homozygous mutations. This causes speculation within the PKD community about whether heterozygous mutations with one mutated copy of the PKD1 or PKD2 gene are sufficient to induce cystogenesis or if two mutated copies are needed. This concept is referred to as the two-hit hypothesis, a somatic mutation (second hit) occurs during the lifetime of the kidney and gives rise to clonal populations of cells that give rise to cysts. The goal of my project is to investigate the two-hit hypothesis and determine if there is cyst formation in the heterozygous organoids. To do so, I use patient-derived hSPC lines modified using CRISPR to express PKD1 or PKD2 mutations. I follow a 21 day differentiation process to produce PKD organoids. I then pick individual organoids to place in suspension for 14 days and track cyst growth by area and amount. If cysts form in heterozygous PKD organoid models at the same rate as the control, it would suggest that heterozygous mutants are unlikely to cause cyst formation, indicating the two-hit hypothesis applies to PKD. Current data collected on mutations in PKD2 have shown heterozygous organoids do not form cysts, however more data is needed to test the PKD1 site. Future applications involve inducing cyst formation in heterozygous mutants using forskolin, an agonist of adenylyl cyclase shown to induce cyst growth. Establishing if heterozygous mutant organoids form cysts has significant implications in ADPKD research by potentially creating a better representation of the disease process, which has been difficult to study in other models.

Type 1 diabetes (T1D) is an autoimmune disease in which the pancreas is unable to produce enough insulin to effectively regulate sugar into the body’s cells. Recent literature suggests that T1D has a prevalence and incidence of 9.5% and 15 per 100,000 people respectively. T1D risk is multifactorial but is heavily dependent on genetics and the environment. Twin studies have shown that although disease risk for the general population is 0.4%, children of diabetic patients are 2 to 9% at risk and identical twins are up to 70% at risk of developing T1D. Genome-wide association studies have shown that many genetic variants associated with T1D risk are found in gene regulatory regions such as enhancer elements. As T1D is an autoimmune disease with a strong genetic component particularly in non-coding, regulatory regions, it follows that thorough genomic profiling of immune cells such as T cells can help identify functional non-coding genetic variants that alter gene regulation for target genes associated with disease risk. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) allows for unbiased identification of cis-regulatory elements (CREs). By utilizing ATAC-seq on various T cell types of both healthy control donors and T1D patients, chromatin accessibility can be collected, and the underlying sequence data can be used to determine allelic differences in transcription factor binding due to T1D-associated genetic variants. I am currently applying ATAC-seq to three T cell subtypes isolated from both donor cohorts. Through these data functional links will be made between non-coding genetic variants and associated target genes to better understand how they impact disease risk in T1D.

Protein kinase R (PKR) is a host antiviral factor that shuts off translation in response to double-stranded RNA that accumulates during viral infection. Many viruses, including cytomegaloviruses (CMVs), encode proteins that inhibit PKR and rescue viral protein synthesis in infected cells. In the case of CMV, the “terminal repeat short 1” (TRS1) protein antagonizes PKR. Analyses have shown that PKR has been evolving under positive selection among primates, likely in order to evade TRS1 and other viral antagonists. Consequently, the ability of TRS1 to block PKR is quite species-specific. For example, the TRS1 homolog in human and Old-World monkeys (OWMs) CMVs only inhibit PKR from great apes and OWMs, respectively. Thus, it is surprising that TRS1 encoded by a New World monkey (NWM) virus, squirrel monkey CMV, antagonizes hominoid and OWM PKRs. I hypothesize that squirrel monkey CMV TRS1 has broad activity against diverse PKRs because it interacts with a conserved domain of PKR that is different from the region bound by TRS1 encoded by human CMV or OWM CMVs. To test this hypothesis, I studied how TRS1 and PKR compete to control protein synthesis in infected cells by using assays of protein synthesis and of replication of wild-type and mutant viruses. Because there has been no study of squirrel monkey PKR (smPKR), I first tested its function using a reporter gene transfection assay and demonstrated that it is active in inhibiting translation, as expected. I cloned smPKR into a doxycycline-inducible lentiviral vector and tested the susceptibility of it and other NWM PKRs to alternative TRS1 genes. By understanding the evolution and molecular mechanisms that have diverged in the ongoing “arms race” between NWMs and their viruses we can reveal insights into the risks and barriers to cross-species transmission of viruses.

Several age-related neurodegenerative diseases include abnormal protein deposition of tau, amyloid-β (Aβ), and TDP-43. The most common dementia, Alzheimer’s Disease (AD), is tauopathic, meaning it is mainly characterized by tau deposition. In neurons, tau normally functions to bind and stabilize microtubules (MTs), proteins made of tubulin dimers that provide neuronal structure and assist in molecular transport along axons. However, in AD, tau becomes hyperphosphorylated, resulting in disassociation from MTs and aggregation in the cell. Previous genetic screening identified several new mutations in genes encoding tubulin proteins that ameliorate the effects of tau toxicity in tauopathy models of Caenorhabditis elegans; however, the various genes confer suppression to varying levels. Given that tubulin genes are expressed differentially in neurons, I hypothesized that the level of gene expression may correlate with the level of tau toxicity suppression for a given gene. To test this, we constructed transgenic C. elegans strains overexpressing mutant tubulin at differing levels. I characterized age-matched worms for tau-induced motility defects and assessed tubulin transgene expression by qPCR, observing that higher levels of mutant tubulin gene expression correlated with higher levels of toxicity suppression. Additionally, since previous work showed that tubulin mutations could confer suppression in a tau-Aβ copathology model, I sought to elucidate whether this result is generalizable to a copathology model with tau and TDP-43. To test this, I generated strains of worms expressing human tau and TDP-43 as well as mutant tubulin, and assessed them for motility defects. I expect that suppression will be conferred in these animals, although not to the same extent as in a model without copathology. These experiments will help increase our understanding of the molecular mechanisms underlying mutant tubulin mediated tau suppression. Additionally, we will gain knowledge about the mechanisms behind tauopathic disease progression in models of pure tauopathy and copathology.

Cachexia is a debilitating syndrome that accelerates muscle and fat wasting, affecting up to 80% of cancer patients with no current effective treatment. This condition is associated with weakness, fatigue, and poor tolerance to chemotherapy. Recently proposed as a therapeutic option due to its anabolic effects on preserving muscle and adipose tissue, ghrelin has been reported to attenuate Lewis Lung Carcinoma (LLC)-induced weight loss and lipolysis, also partially regulated by its only known receptor growth hormone secretagogue receptor (GHSR-1a). Tumor-induced lipolysis is associated with an alteration of substrate utilization. However, the extent to which these effects of ghrelin relate to substrate utilization (lipid oxidation and carbohydrate oxidation, LO and COX) remains unclear. This project seeks to determine if ghrelin’s effect on LO and COX in the LLC-induced cachexia model is dependent on GHSR-1a. Adult male C57BL/6J GHSR-1a knockout (KO) and wild-type (WT) mice were treated with or without LLC tumor, then injected with vehicle or ghrelin (0.8 mg/kg). Metabolic parameters were evaluated by the Comprehensive Lab Animal Monitoring System, in which I analyzed LO and COX (calculated mean values and performed statistics). Tumor implantation led to an increase in LO and a decrease in COX in tumor-bearing mice. GHSR-1a KO mice had a greater increase in LO compared to WT, highlighting the receptor’s essential role in maintaining normal levels of LO during cachexia, with no genotype effect for COX. Ghrelin did not prevent the LLC-induced response regardless of GHSR-1a expression, hence its mitigating effects for lipolysis in cachexia are not dependent on the regulation of LO or COX. In conclusion, GHSR-1a plays a role in modulating LO in tumor-induced cachexia, and these effects are independent of ghrelin. More studies are needed to further characterize the pathways involved, including alternative receptors of ghrelin or its adjacent mechanisms.

Liver transplantation (LT) is the only curative treatment for advanced chronic hepatitis B (CHB), however, these patients are at significantly higher risk of developing chronic kidney disease (CKD) post-LT. Therefore, it is important to understand the renal impacts of life-long antiviral therapies for CHB, commonly including tenofovir alafenamide (TAF), tenofovir disoproxil fumarate (TDF), and entecavir (ETV). To study this, I conducted a retrospective cohort study in collaboration with five multinational LT centers of 298 LT recipients who received TAF (n=112), TDF (n=51), or ETV (n=135) monotherapy for at least 12 months post-transplant. To measure changes in renal function, I analyzed CKD stage and estimated mean glomerular filtration rate (eGFR, mL/min/1.73 m2) data, where higher eGFR indicates better renal function. I found that at baseline, TAF patients compared to TDF and ETV patients were older (P=0.02), had higher rates of hypertension (P=0.048), and had lowest eGFR (P=0.01). However, from baseline to the 24th month of follow-up, the proportions of patients with normal renal function (CKD stage 1) and mild renal impairment (stage 2) remained stable for the TAF and TDF groups but significantly shifted towards poorer renal function in the ETV group (CKD stage 1: 35.56% to 18.18%, P=0.002; stage 2: 35.29% to 49.59%, P=0.006). TAF patients also had the smallest decline in mean eGFR from baseline to the 24th month of follow-up (1.46 vs. 2.97 (TDF) vs. 4.57 (ETV)). In conclusion, I found that patients treated with TAF had baseline characteristics correlated with poorer renal function, however, their CKD stages remained stable and had the smallest eGFR decline throughout follow-up. These trends reveal an interest in further investigating the renal impacts of TAF treatment in post-LT patients.

Insulin release normally leads to the reduction in blood glucose levels; type 1 diabetes mellitus is a disease caused by a lack of insulin production by the pancreas that affects millions of people around the world. Diabetic patients are tasked with injecting insulin daily to support optimal blood sugar levels. Recently, a gastric-resistant fast dissolving tablet form of insulin has been synthesized, which can ease the process of intaking insulin. The purposes of our in vivo studies are to test the efficacy of the novel oral insulin formulation in lowering blood glucose levels and to compare the blood glucose lowering effects of oral versus conventional subcutaneous insulin. We hypothesized that the oral administration of the novel insulin will reduce blood glucose levels more efficiently than both oral and subcutaneous administrations of conventional, non-encapsulated insulin since the insulin tablet mimics the pathway of natural insulin release within the body. We used a rodent model using a sample size of 10 mice to test the pharmacodynamic effects of the novel insulin - in other words, how the formulation affects the body. To do so, I compared the efficacy of the novel insulin to conventional insulin through insulin tolerance tests using intraperitoneal injections of the administration of novel insulin of 1 U/kg body weight. Furthermore, I performed glucose tolerance tests using an oral gavage to analyze the time course for normalizing blood glucose levels. We expect preliminary results to show a statistically significant difference in effectively lowering blood glucose levels in response to the novel formulation relative to the conventional insulin. The results of these studies are crucial to determining proper dosing concentrations and viable insulin administration schedules that can aid prospective human clinical trials.

Glioblastoma multiforme (GBM) is known as the most aggressive brain tumor and almost always lethal. The DNA methylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter gene has been extensively used as a GBM biomarker to predict prognosis and stratify patients for treatments. Previous research has shown that GBM features exhibit significant molecular heterogeneity within a tumor, which has forced the utilization of multiple, rather than single, biopsies to optimize potential therapies. Therefore, the goal of this project is to define the scope of intratumor heterogeneity of MGMT methylation in GBM and its relation to tumor histology to determine sampling biopsy needs. The lab has developed high throughput systems to preserve and sample tissue in frozen state using CryoGrid-CryoCore system and to prepare DNA using PIXUL multi-sample sonicator. To assay epigenetic markers, including DNA-methylation, the system incorporates matrix methylated DNA immunoprecipitation (MeDIP) and quantitative PCR (qPCR). We section dozens of frozen GBM tumors and use CryoGrid-PIXUL-Matrix-MeDIP-qPCR system to assess intratumor MGMT methylation heterogeneity. Thus far, our results suggest that low level of intratumor MGMT methylation heterogeneity and sampling GBM tumor in three different regions will provide clinicians with reliable information regarding prognosis and treatment of this deadly cancer.

Cardiomyopathies are diseases of the heart characterized by structurally and functionally abnormal cardiac tissue and can be caused by non-genetic or genetic causes. Genetic cardiomyopathies are the most common genetic cardiac condition, affecting 1 in 250 to 500. The two most common types of genetic cardiomyopathies are hypertrophic (HCM) and dilated (DCM). HCM is characterized by a thickening of the heart muscle. This thickening can lead to a blockage in the blood flow and cardiac relaxation abnormalities. DCM is pathologized by a weakening in the cardiac muscle, leading to a lengthening and thinning in the muscle. My research focuses on determining human specific mechanisms of DCM and HCM, specifically on determining the early developmental phenotypes of the cells that lead to downstream pathologies. I particularly emphasize how changes in sarcomere function lead to HCM and DCM using human induced pluripotent stem cell cardiomyocytes (hiPSC-CM). We have shown that sarcomeric mutations alter the amount of tension integrated over time (TTI) and those variations in TTI are predictive of HCM and DCM. I am generating two mutant hiPSC-CM lines, L48Q and I61Q, using CRISPR/Cas9. These mutations are both in the sarcomere, more specifically in cardiac troponin C (cTnC). They alter the calcium binding properties of cTnC. I have optimized the polymerase chain reactions in order to make the sequencing data clean for validation. I have generated the I61Q line and am working on the L48Q line. After validating the lines, I will differentiate them to cardiomyocytes in order to study the cellular mechanisms involved. I will then use IonOptix to test early vs. late calcium transience, cell contractility, and cell size. There are currently no treatments that address the contractile abnormalities present in HCM or DCM. My research will allow for greater understanding of these mechanisms which will inform potential therapies.

Paraoxonases (PONs) are a family of three closely related genes found on the long arm of chromosome 7. The genes encode PON1, PON2, and PON3, which are primarily involved in metabolizing oxidized lipids and modulating oxidative stress. However, each of the PON enzymes are involved in important secondary reactions. PON2 is an intracellular enzyme localized in the mitochondria that plays a vital role in modulating oxidative stress and inactivating microbial quorum sensing factors. Individuals with PON2 deficiencies are sensitive to oxidative stress. It may be possible to restore PON2 function by creating an injectable protein for individuals with a PON2 deficiency. Our goal is to actively express recombinant PON2 in an E. coli expression system and inject PON2 into PON2 knockout mice to determine if PON2 function can be restored. To express PON2 in E. coli we designed a synthetic DNA sequence by removing the transmembrane sequence of PON2 and replacing it with the signaling sequence from PON1, which facilitates the purification of the chimeric protein. We transformed the synthetic PON1/PON2 plasmid in E. coli and are currently performing gel electrophoresis and activity assays to analyze the expression of PON2. If we see protein expression as expected, then we will purify the recombinant PON2 for injection using a histidine tag that we added to the end of the protein coding sequence in the construct. The histidine tag allows for single-step purification via affinity chromatography, which we will then inject into PON2 knockout mice to observe their response to oxidative stress. Findings from this experiment will allow for further understanding of PON2 function and its restoration via an injectable protein, as well establishing that E. coli expression systems can be used as a more cost-effective method for pursuing further PON2 related research.

In the cold, the energy demands for heat production increase to defend core body temperature. A hyperphagic response must balance these energy costs, or body weight cannot be maintained. However, this hyperphagia may contribute to the problem of obesity in our general population, thus raising the importance of understanding the underlying neuronal mechanisms. We recently found in mouse models that agouti-related peptide (AgRP) expressing neurons located in the arcuate nucleus of the hypothalamus (ARC) are thermoresponsive and required for cold-induced hyperphagia. However, the afferent neurocircuit capable of driving AgRP neuron activity and modifying food intake drive in the cold is unknown. I recently assisted in identifying a novel population of tyrosine hydroxylase-expressing neurons located in the rostral paraventricular nucleus of the hypothalamus (PVH-TH). Using fiber photometry, we found these neurons respond similarly to AgRP neurons, increasing their activity with cold and reducing their activity in response to food-related cues. I found that hemogenetic activation of these rostral PVH-TH neurons mimics the effect of cold exposure on energy intake and elicits a modest thermogenic response. This study provides evidence for a link between thermoregulatory and food intake neurocircuitry in the mouse, setting the stage for further investigations into the role of ambient temperature on food intake drive. Because these systems are uncoupled in the setting of obesity, our findings may provide future therapeutic options for the treatment or prevention of obesity.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 infection. COVID-19 is associated with higher risk of new-onset diabetes and metabolic complications of diabetes, which may occur via injury to insulin-producing islet β cells due to direct SARS-CoV-2 entry. While the canonical pathway of viral entry via human angiotensin converting enzyme 2 (ACE2) has been established as a major route of SARS-CoV-2 infection in the lung, expression of ACE2 has not been unequivocally demonstrated in beta cells. It is therefore important to understand how other proteins known to be highly expressed in pancreatic endocrine cells may be involved in SARS-CoV-2 entry. Glucose regulatory protein 78 (GRP78), also known as binding immunoglobulin protein (BiP), is an endoplasmic reticulum (ER) chaperone. Under conditions of ER stress, as induced by SARS-CoV-2 infection, GRP78 translocates from the ER membrane to the plasma membrane, where it has been shown to bind to SARS-CoV-2-spike in vitro. Previous studies have found GRP78 to aid the entry of other coronaviruses. As GRP78 is expressed in β cells, we investigate GRP78-mediated SARS-CoV-2 entry into β cells using a mouse β cell line, wherein mouse ACE2 is unable to bind SARS-CoV-2-spike. To promote GRP78 translocation to the plasma membrane, ER stress is induced using thapsigargin or cytokine treatment. Verification studies involve validating the upregulation of GRP78 at the mRNA level and in plasma membrane fractions. A cell fractionation protocol has been adapted to isolate plasma membrane protein from other cellular fractions. Further experiments will involve the use of a pseudovirus system to investigate SARS-CoV-2 entry under conditions of GRP78 overexpression and knockdown. Studying SARS-CoV-2 entry into β cells may illuminate possible therapeutic strategies to protect islets from the deleterious effects of COVID-19, including the development of diabetes.

Fanconi anemia is a serious genetic blood disorder caused by mutation in the FANC genes, responsible for the regulation of DNA interstrand crosslink repair. Failure of this mechanism results in chromosomal instability and is extremely damaging. Patients are also at a greatly increased risk for malignant cancers such as acute myeloid leukemia. The goal of the project is to develop novel methods to treat Fanconi anemia using gene therapy-based vectors, including a lentiviral vector containing an shRNA CD33 knockdown that confers selective protection, and to eliminate dangerous residual Fanconi anemia cells following treatment. Through in vitro validation, I will test several therapeutic vectors that provide a functional copy of the gene for correction of the FANCA defect and protection of corrected cells from drugs that target CD33, which are commonly used to treat secondary myeloid malignancies that often develop in Fanconi anemia patients. Then, using a FANCA -/- mouse model, I will use in vivo tests to determine if vectors can rescue the FANCA deficiency and provide selective protection using flow cytometry, cell and colony assays, and qPCR. I identify the more efficient lentiviral vectors for further tests. Together, these experiments are crucial in the development of a clinically viable blood stem cell-based therapy for Fanconi anemia that protects patients from the high risk of secondary malignancies such as acute myeloid leukemia that are so prevalent.

Syphilis is a chronic sexually transmitted infection that is caused by the spirochete bacterium Treponema pallidum subsp. pallidum (T pallidum). Although syphilis is mistakenly believed to be a disease of the past, it is actually still endemic in low and middle-income countries, and it has been steadily resurgent in high-income nations, including the USA, for the past 20 years. An effective syphilis vaccine would greatly help curtail syphilis spread. Our current understanding of syphilis pathogenesis suggests that for a syphilis vaccine to be effective, it must induce antibodies able to opsonize antigens on T. pallidum surface to induce pathogen ingestion by macrophages. To this end, we used three T. pallidum putative surface antigens as vaccine candidates, which belong to the T. pallidum repeat (Tpr) protein family, specifically TprC, TprK, and TprD2. Once synthesized as recombinant antigens, New Zealand White (NZW) rabbits were immunized every three weeks with a total of five injections, and then challenged with infectious T. pallidum on their shaved backs. After the challenge, the efficacy of the vaccine candidate was tested by monitoring several correlates of protection, including lesion development and treponemal burden within the injection sites. Immunization with the TprC and TprK antigens induced attenuated lesions and reduced treponema burden, but not immunization with TprD2. Although at the end of the experiment all the rabbits were positive for syphilis infection based on serology, we demonstrated that TprC and TprK were effective in inducing partial protection, and therefore should be considered as vaccine candidates for this serious infection.

Tuberculosis (TB), a respiratory disease caused by Mycobacterium tuberculosis (Mtb) bacteria, continues to be a leading cause of death worldwide. There is an overwhelming need to better understand protective immunity and develop an improved vaccine due to variable efficacy in preventing infection with the current TB vaccine, Bacillus Calmette–Guérin. T cells are required for the protective immune response to Mtb infection. γδ T cells are a subset of T cells known to be involved in Mtb protection, but the mechanisms by which they control Mtb remain poorly understood. However, γδ T cells are a major source of the cytokine IL-17, which promotes lung inflammation, and recruits cells for immune response, suggesting that IL-17 may contribute to γδ T cell control of Mtb. I characterized the T cell secretion of IL-17 in a cohort of South African adolescents that have latent Tb infection or are TB uninfected. I sorted γδ T cells and É‘êžµ T cells from sample PBMCs and stimulated these cells with Mtb antigens to measure the secretion of IL-17 via an enzyme-linked immunosorbent assay. I hypothesized that T cells taken from latently infected individuals would secrete greater amounts of IL-17 compared to those that are not infected due to a preexposure to Mtb antigens. Additionally, I predicted that γδ T cells from latently infected individuals would secrete a higher proportion of IL-17 compared to É‘êžµ T cells. Investigating the IL-17 secretion by T cells can potentially provide us with a better understanding of the role that IL-17 producing T cells play in protective immunity and inform vaccine development.

Human Herpesvirus-8 (HHV-8) causes Kaposi’s sarcoma (KS)-- a cancer of cells that line lymph or blood vessels. Individuals whose T cells have been compromised by HIV are particularly at risk for KS. The disease remains endemic in many parts of sub-Saharan Africa, making KS a leading cause of cancer death in Uganda. While HHV-8 has been known to cause KS since 1994, and T-cells that can recognize HHV-8 are likely to be critical for control of KS, there is little known about the specific parts of the virus recognized by T-cells. Our goal is to ultimately determine the antigenic targets of HHV-8-specific T-cells. Because recovery of live T-cells from biopsies is challenging, we are re-creating “artificial T-cells” with candidate T-cell receptor (TCR) sequences obtained from KS lesion biopsies from Uganda. These candidate TCRs will be queried for reactivity to HHV-8. To do this, we are generating a set of every known HHV-8 protein by moving cloned DNA expressing these proteins into a specific plasmid that is useful for these T-cell studies. To create artificial T-cells, we are cloning candidate TCRs into lentiviral vectors which allow us to force the TCRs to be expressed in these cells and fluoresce green if the cells recognize a viral protein through their TCRs. These artificial T-cells are then screened against every HHV-8 protein to find which protein they recognize. Currently, flow cytometry results demonstrate adequate expression of candidate TCRs by artificial T-cells, and the HHV-8 protein set is >90% complete. Ultimately, we hope to determine HHV-8 proteins that are recognized by HHV-8-specific T-cells in order to better understand which parts of the virus are targeted by these T-cells and to design T cell or vaccine therapies to treat persons with KS.

Atherosclerosis is the build-up of cholesterol, fats and other substances in artery walls. These builds-ups (plaques) can cause vessel blockages, leading to heart attacks and strokes. Removal of cholesterol from plaques is facilitated by the physiologic process of reverse cholesterol transport (RCT). In RCT, apolipoprotein AI (apoAI) protein removes cholesterol from vascular tissue, forming high-density lipoprotein (HDL) that is transported to the liver for excretion. Gene-therapy strategies that use vectors to increase apoAI expression in arteries slow and reverse atherosclerosis in rabbits. However, because apoAI has multiple activities including reducing inflammation, it is unclear whether vector-derived apoAI reduces atherosclerosis by contributing to RCT. To determine whether apoAI gene therapy contributes to RCT, I am developing a method that can track HDL that contains vector-derived apoAI in vivo. If we can detect vector-derived apoAI/HDL in treated arteries as well as plasma and liver, we will conclude that vector-derived apoAI contributes to RCT. I am first developing methods that purify and detect endogenous apoAI/HDL; these methods will be applied to detect vector-derived apoAI/HDL (the vector-derived apoAI will have a molecular tag). I used density-gradient ultracentrifugation to isolate HDL from pulverized rabbit tissue and plasma, then immunoblotting to detect apoAI/HDL. I detected abundant apoAI/HDL in plasma, but not in aorta and liver. Immunoblots of artery extracts (before ultracentrifugation) detect apoAI, suggesting that apoAI is lost during ultracentrifugation. I am testing whether other methods can purify apoAI/HDL from aorta and liver (e.g. affinity chromatography to isolate apoAI from tissue extracts, followed by size-exclusion chromatography to isolate apoAI/HDL). After I develop a method to isolate apoAI/HDL from tissue, we will treat rabbit arteries with a tagged apoAI vector and measure tagged apoAI/HDL in arteries, plasma, and liver. Verifying how vector-derived apoAI works will help us to improve its atheroprotective effects.

The brain maintains body weight homeostasis via a tightly regulated neuronal circuitry. Microglia, the innate immune cells of the brain, elicit an inflammatory response that triggers increased food intake and weight gain on a high fat diet. We are curious how microglia regulate the neuronal circuitry to affect food intake and body weight. We have developed a chemogenetic mouse model that expresses a modified Gs-protein-coupled DREADD (Designer Receptor Exclusively Activated by Designer Drugs) selectively on microglia. Administration of the ligand Clozapine-N-Oxide (CNO) activates the cyclic AMP signaling cascade in microglia. We have found that CNO administration for three days increases the cytokine IL-1𝞫, and reduces chemotactic signals (P2RY12 and CCL3) and Agouti-Related Peptide (AgRP). AgRP is produced from neurons in the hypothalamus and is responsible for feelings of hunger. I hypothesize that microglial Gs-DREADD activation suppresses AgRP signaling and reduces food intake and body weight. To test this hypothesis, mice expressing the microglial Gs-DREADD (MG Gs-DREADD+) and control littermates (MG Gs-DREADD-) will receive daily intraperitoneal administration of CNO (1 mg/kg), and will be monitored for food intake and body weight for a week. Mice will then be placed on a high fat diet (60% kcal obtained from fat) under daily CNO administration, and will be monitored for food intake and body weight gain for 4 weeks. I hypothesize that MG Gs-DREADD+ mice will show reduced food intake and weight gain on a HFD when compared to MG Gs-DREADD- mice. At the end of the study, I will examine changes in inflammatory mediators and neuropeptides in the hypothalamus of the mice. Overall, this study will help elucidate how microglia alter hunger and satiety signals in the brain to regulate appetite and body weight. Moreover, the ability to modify these signals can help individuals manage their weight and prevent obesity.
 

Avoidable prolonged hospital stays can decrease bed availability for new patients and place patients at risk for adverse events, and be costly for hospital finances. There are few published studies that cover data of patients throughout an entire hospital; most only cover data from a specific service line. Our study aims to review patient data throughout an entire hospital to assess common barriers leading to these avoidable prolonged stays. In our institution, the care management department meets on a weekly basis to review patients who meet the crieteria of having avoidable days. We retrospectively reviewed notes from these weekly meetings and extracted common barriers, which we then organized into categories for comparison. Our study included patients discharged from August 2019 to February 2020, with any avoidable days and 21 or more hospital days. There were 180 encounters that met our criteria. The most common service lines represented are Medicine and Surgery, while the most common discharge locations are SNF and Home. The median for the total legnth of stay was 42 days, while the median for avoidable days was 17.5 days. We identified 21 common barriers. The most common discharge barriers identified are Funding, Long Term Care, COPES, Guardianship, Substance Use, Homelessness, and Care Needs too Great for SNF. In our hospital-wide study, we identified that avoidable days are found across inpatient services and represent a heterogeneous mix of discharge barriers. Further studies should investigate relationships between discharge barriers and whether any patterns exist in patients with avoidable days in service lines. 

In the United States, one-third of the adult population is considered obese. Although excessive food intake leads to obesity, a return to normal body weight is extremely difficult once established. A growing body of evidence finds that changes in the brain prevent a return to normal body weight. In particular, Agouti-related peptide (AgRP)-expressing neurons, which drive feeding and feeding-related behaviors, show blunted activity in response to food and hormonal cues in obese mice. Using a mouse model, our group found that sensed ambient temperature regulates AgRP neuron activity, such that cold increases AgRP neuron activity, and this increase in activity drives food intake, but this temperature sensitivity is lost in the obese mouse model. However, how AgRP neurons receive thermal information is not understood—owing to the speed with which AgRP neuron activity changes in response to cold sensation, thermal information is most likely relayed by an afferent circuit. Here, I report the identification of a novel population of tyrosine hydroxylase (TH)-expressing neurons located in the rostral paraventricular nucleus of the hypothalamus (PVHTH neurons) as candidate upstream mediators of cold-induced AgRP neuron activation. As evidenced by the expression of the immediate early gene, Fos, cold activates this population similarly to AgRP neurons, and these neurons send projections to the arcuate nucleus of the hypothalamus (ARC), where AgRP neurons are located. I hypothesized that activation of this population, as part of a larger thermoregulatory neurocircuitry, would shift the preferred ambient temperature of mice such that they prefer warmer temperatures, as well as increase food intake to levels seen in mice housed in the cold. My findings underscore the link between thermoregulation and energy homeostasis and begin to define a circuit which, if disrupted, might underlie the perseverance of obesity.

Prostate cancer is the second leading cause of cancer death among men in the United States and its detrimental form, known as castration-resistant prostate cancer (CRPC), currently has no cure. The importance of cancer metabolism has been recognized as a potential target of anti-CRPC therapy. The Plymate Lab recently developed compounds known as selective glycolytic inhibitors (SGIs) that target glycolysis in selective types of cancers including CRPC. Specifically, SGI’s block the first step of glycolysis, in which glucose phosphorylation is catalyzed by hexokinases. Nevertheless, SGI proved not to be a traditional catalytic inhibitor. HK1 and HK2 are major hexokinases in prostate and other cells and their mitochondrial localization is pivotal for their activities. The goal of my project is to test the hypothesis that SGIs affect mitochondrial localization of HK1 and HK2. I used immunofluorescence techniques to examine the physical association between the hexokinases and mitochondria. To do so, I treated a prostate cancer cell line, LNCaP, with SGIs for 30 minutes, which is the amount of time I found the anti-glycolytic effect of SGIs to be evident. Upon doing so, I did not see any significant changes in subcellular localization of HK1 and HK2 between treated and non-treated groups. The majority of HK1 and HK2 were associated with mitochondria, which were fluorescently visualized by MitoTracker Red CMXRos, which labels mitochondria with red fluorescence. It has been reported that the association of mitochondria with hexokinases largely relies on its ability to bind to VDAC1. Thus, I am currently carrying out co-immunoprecipitation assay to assess physical association between HKs and VDAC1. This study will help determine whether SGIs display their anti-glycolytic activity by altering subcellular localization of hexokinases.

Tuberculosis (TB) is one of the leading causes of death from infectious disease worldwide. Macrophages are the primary replicative niche and provide critical host defense against Mycobacterium tuberculosis (Mtb), the causative organism. A TOLLIP deficiency is associated with an increased risk of TB in human studies and mouse models. TOLLIP is a ubiquitin-binding protein that interacts with toll-like receptors (TLR) involved in modulating inflammatory signaling. TOLLIP has four critical domains: a ubiquitin-binding CUE domain, a membrane binding C2 domain, an autophagosome interacting motif (AIM), and a Tom1-binding domain that participates in endosomal sorting. However, the domains required for TOLLIP’s immune regulatory function during Mtb infection in macrophages are uncertain. To understand the role of selected domains on TOLLIP’s function, we are developing lentiviral vectors to insert TOLLIP with selected protein domains deleted, along with green fluorescent protein (GFP) to tag transduced cells. We measured viral load and transduction efficiency in HEK cells by analyzing GFP fluorescence through microscopy and flow cytometry. We are currently transducing TOLLIP fragments, whole TOLLIP, and control genes into macrophages lacking the TOLLIP gene. TOLLIP (-/-) bone-marrow derived macrophages (BMDM) were infected with lentivirus after 3 days and stimulated with LPS (10ng/ml) after 5 days. Viral supernatants were collected to evaluate TOLLIP gene expression through fluorescent microscopy. The approaches developed in this project will provide the foundation for understanding the critical structural elements of the TOLLIP protein and their role in preventing TB disease.

Following allogenic hematopoietic cell transplantation (allo-HCT), patients are at risk of developing graft-versus-host disease (GvHD) in which donor T-cells attack the recipient’s healthy tissues, including in the gut leading to inflammation, diarrhea, and sometimes death. Low gut bacterial diversity in the host has been associated with GvHD severity. Anaerobic bacteria in the Lachnospiraceae family, specifically Blautia species, have been associated with reduced GvHD related mortality. Using family and genus specific qPCR assays, we quantified the bacterial concentrations of Lachnospiraceae and Blautia species pre- and post-transplant in patients. We extracted DNA from stool samples of 306 HCT patients. Samples were collected pre-transplant and post-transplant at day 30 and day 60. GvHD gut stage was graded 0-1 (none-mild), 2-4 (moderate-severe). Quantitative PCR assays were designed using primers targeting specific regions of the bacterial 16S rRNA gene. Amplicon specificity was confirmed using post-run melt curve analysis. There was a significantly higher (p=0.016) concentration of Lachnospiraceae in patients presenting none-mild GvHD (6.78x10⁷ copies per swab) versus moderate-severe gut GvHD (1.84x10⁷) at day 60. A similar association for Lachnospiraceae was trending (p=0.088) at day 30. In addition, there was significantly higher concentrations of Blautia species in patients with none-mild (3.33x10⁶ at d30, 1.05x10⁷ at d60) versus moderate-severe gut GvHD (1.35x10⁵ at d30, 1.41x10⁶ at d60) at both day 30 (p=0.026) and day 60 (p=0.014) post allo-HCT. There were lower concentrations of both Lachnospiraceae (1.89x10⁷) and Blautia (2.30x10⁶), regardless of GvHD stage, at day 30 versus pre-transplant (p<0.0001) or day 60 (p<0.0001), likely reflecting the impact of antibiotic treatment during neutropenia immediately following allo-HCT. Higher concentrations of Lachnospiraceae and Blautia in the gut following transplant were associated with less severe gut GvHD; these bacteria could be markers or drivers of less severe GvHD.