HIV establishes latency in long-lived, proliferative memory CD4+ T cells, allowing the virus to evade immune clearance. The “shock and kill” strategy seeks to reactivate latent HIV using latency-reversing agents (LRAs), enabling immune-mediated or therapeutic clearance. However, LRAs are problematic due to toxicity and lack of specificity and efficiency. These limitations underscore the need to understand LRA mechanisms to develop more effective therapies. Phosphorylation is a key regulator of transcriptional and nuclear events, yet how LRAs reshape them is poorly understood. This project examines phosphorylation dynamics during early HIV reactivation. Using the J-Lat 10.6 cell lines and three LRAs (SAHA, PMA, prostratin), we performed global proteomics and phosphoproteomics via LC-MS/MS, identifying over 150,000 phosphopeptides and 8,000 phosphoproteins. We found that LRAs induce extensive host phosphoproteome remodeling, and they converge on the regulation of key nuclear complexes through different phosphosites, such as the Nuclear Factor kappa B (NF-κB) complex. Future work includes using size exclusion chromatography-mass spectrometry (SEC-MS) to capture phosphorylation-dependent complex remodeling and affinity purification-mass spectrometry (AP-MS) to map interactomes of key phosphoproteins. These would pinpoint specific mechanisms and identify shared host factors to improve and design therapeutic strategies for HIV cure.

Alphaviruses are arthropod-borne arthritogenic and encephalitic viruses containing a positive sense single-stranded RNA genome. When detected by the host innate immune system, the interferon (IFN) response is activated leading to increased production of interferon-stimulated genes (ISGs). Zinc-finger antiviral protein (ZAP) is an RNA binding protein and CG dinucleotide sensing ISG with broad yet specific antiviral activity, as it restricts certain viruses from multiple, distantly related families. Sindbis virus (SINV) is among the viruses ZAP potently restricts through the binding of viral RNA, while chikungunya virus (CHIKV) effectively resists ZAP. Using enhanced cross-linking immunoprecipitation and sequencing (eCLIP-seq), we found ZAP binds SINV RNA in the non-structural protein 1 (nsP1) gene and the 3’ untranslated region (3’ UTR), but no convincing binding sites along CHIKV RNA. Through Topoisomerase (TOPO) cloning and High-Fidelity DNA Assembly (HiFi assembly), we generated chimeric viruses swapping these ZAP binding regions of SINV into CHIKV and vice versa. To determine the functional consequences of ZAP binding and examine the determinants of its differential sensitivity, we infected ZAP knockout 293T cells with doxycycline-inducible ZAP overexpression, with both parental and chimeric viruses. Elucidating the poorly understood mechanism of ZAP’s ability to inhibit a broad, yet specific range of alphaviruses, presents a key opportunity to inform the development of novel therapeutic options against alphaviruses.

Bladder cancer remains a significant clinical challenge due to high recurrence rates and the limited efficacy of current treatments, including Bacillus Calmette-Guerin (BCG) immunotherapy. We have developed a chimeric antigen receptor (CAR) T cell therapy targeting the NECTIN4 surface antigen as a promising alternative. Yet, its success in solid tumors is limited by the immunosuppressive tumor microenvironment and reduced cytotoxicity in urine. To overcome this, we investigated a novel approach to enhance the potency of NECTIN4 CAR T cells with vault nanocapsules, which are naturally occurring and non-immunogenic ribonucleoprotein particles, to deliver therapeutic cytokines to the tumor area via intravesical installation. We evaluated whether cytokine-packaged vaults enhance CAR T cell efficacy using in vitro uptake assays and co-culture systems with human and mouse bladder cancer cell lines (RT112, UMUC9, MB49). Flow cytometry showed efficient vault uptake by both tumor cells and CAR T cells. Ongoing co-culture studies assess whether vaults containing IL-2, IL-15, or CCL-21 improve CAR T cell cytotoxicity. Preliminary in vivo studies in orthotopic mouse models demonstrate successful vault uptake in bladder tumors, supporting intravesical delivery as a viable strategy. These findings suggest that vault-mediated cytokine delivery can enhance CAR T cell function and provide a more targeted therapy with reduced systemic toxicity and prevention of recurrence.

Hypoxemia refers to low blood oxygen levels and is correlated with cognitive impairment (CI). Hypoxemia-induced CI can negatively affect military aviation. Thus, it is essential to find ways to increase the resistance against hypoxia-induced CI. The gut microbiome consists of trillions of microbes that can be changed through dietary intervention. Microbiome-depleted mice exhibit CI, highlighting that the microbiome is protective against CI. In normoxia conditions, a ketogenic diet intervention reduces CI. However, a ketogenic diet enhances CI under hypoxia. Thus, we must identify treatments to protect against hypoxia-induced CI. In humans, a microbiota-accessible carbohydrate and polyphenols (MACP) diet reduces CI under hypoxia. This project aimed to determine whether MACP remodeling of the gut microbiome drives the protection against hypoxia-induced CI. Human participants were supplemented with a placebo then MACPs for 12 days each. Fecal samples were collected after each dietary treatment and transplanted into mice. Mice went through 6-hour hypoxia or normoxia treatment for 5 days. CI was measured with the Barnes maze test. We found that the microbiome is linked to the protective effect of MACP supplementation on CI and this protective response is dependent on the individual. This research facilitates the development of diet and microbiome-based supplements that reduce hypoxia-induced CI.

Wound healing is a tightly regulated process involving coordination between numerous factors, and fibroblasts play a central role. Dysregulation of a component such as the Col5a1 gene, in an in vivo model, has been shown to impair wound healing and prolong inflammation, partially corrected by adding WT fibroblasts. This study explored an in vitro model of wound healing using WT and Col5a1-deficient mouse fibroblasts to investigate prolonged inflammatory responses and if it influences fibroblast activation, migration, and ECM remodeling. Primary fibroblasts were isolated and cultured. Experimental conditions included a mechanical scratch assay, serum modulation, lipopolysaccharide stimulation, and TGF-β stimulation to mimic a wound microenvironment. Cells were analyzed across time points using qPCR to assess expression of inflammatory cytokines, fibrotic markers, and matrix remodeling enzymes, including IL4, IL6, CCL2, TGFB1, and ACTA2 etc. Results indicate time-dependent upregulation of genes associated with inflammation, fibroblast activation, and matrix remodeling, supporting the hypothesis that fibroblasts adopt a repair-associated phenotype following injury. This project establishes an initial Col5a1-deficient fibroblast-based scratch assay model as a platform for studying stromal contributions to wound healing and insight into how inflammatory signaling regulates tissue repair. This is significant in informing therapeutic strategies linked to wound healing and fibrosis.