Purdue University

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Opportunistic fungal infection of immune-compromised individuals is an escalating world health problem, recently highlighted in a report from the World Health Organization. Lethal outbreaks of multi drug-resistant Candida auris in hospitals and the rise of drug resistance in normally benign commensal fungal species like Candida glabrata highlight the severity of the problem. Even severe COVID-19 cases facilitate secondary infection by fungal pathogens like Aspergillus and Candida that can be lethal. Current treatment options for fungal infections are limited to a few antifungal drug classes that are becoming increasingly ineffective. There is a pressing need for new molecular targets for antifungal development to deal with drug-resistant pathogens. Our central objective is to establish auxin-inducible degradation (AID) technology in Candida pathogens to enable functional studies of virulence and drug resistance factors and as a tool to facilitate antifungal target validation in the early stages of antifungal drug discovery. AID provides rapid and specific depletion of target proteins of interest and has key advantages over other common methods for protein functional characterization. In Aim 1 we will engineer molecular biology reagents and strains, and establish protocols, to validate and implement a modified AID system in C. albicans, C. glabrata, and C. auris. Our novel system should be applicable in any strain, including clinical isolates, of Candida pathogen species. Validation experiments will use novel virulence and drug resistance factors identified in our labs. In Aim 2 we will combine AID technology in Candida species with two common animal infection models, Galleria mellonella (waxworm) larvae and immunosuppressed mice, to create systems for early target validation and in vivo simulation of drug effects on pathogenesis. These systems will also be applied to our novel candidate antifungal targets. AID is a powerful functional genomics tool that will enable new research opportunities in fungal pathogens. Reagents and protocols established during the project will be made available to the research community, and the work will establish a blueprint for expanding AID use to other diverse fungal pathogens. Overall, this technological platform will address the pressing need for identification and validation of viable new targets for antifungal therapeutic development.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY My long-term career goal is to do hypothesis-driven research as an independent investigator at a university spanning basic and clinical immunology in inflammatory bowel diseases (IBD). In order to achieve this goal, I have put together an advisory team composed of Dr. Matthew Olson (sponsor), Dr. Timothy Ratliff (co-sponsor), Dr. Tzu-wen Cross (collaborator), Dr. Majid Kazemian (collaborator), Dr. Shihuan Kuang, and Dr. Wayne Campbell to provide technical training for in vitro cell culture techniques, in vivo mouse models of intestinal disease, and gnotobiotic training, as well as other professional development skills in scientific writing and communication. My preliminary work outlined in this proposal has demonstrated that mice with conventional T cell-specific deletions in two important T cell transcription factors, STAT3 and BATF, developed an aggressive spontaneous colitis that was marked by a dysregulated microbiota and elevated numbers of γδ T cells in the intestines similar to what is observed in human patients. Given that the current treatment options available for IBD are not fully effective in long term treatment, my data indicate that disrupting this STAT3/BATF-axis through targeting the dysregulated gut microbiota or γδ T cells may represent a novel therapeutic strategy. To further understand this complex relationship between dysregulated gut microbiota and non-conventional γδ T cells in IBD-like colitis, this proposal will address the following: (1) Elucidate the role of the microbiota in regulating intestinal γδ T cell homeostasis during intestinal disease. 16S rRNA sequencing will be used to define shifts in the microbial community in each mouse genotype. To determine if this microbiota is required and sufficient to induce γδ T cell responses and disease we will treat animals with antibiotics and perform fecal microbiota transfers into germ-free animals, respectively. (2) Determine the role of γδ T cells in driving IBD. Therapeutically, I will treat mice with monoclonal antibodies that block γδ T cell receptor activation and monitor its impact on inflammatory responses in the intestines and clinical disease development. In order to determine if γδ T cells can confer disease, I will isolate γδ T cells from Stat3fl/flBatffl/flCd4Cre+ mice and transfer them into colitis susceptible (Rag-/-) mice. The results of this proposal will further elucidate the role of γδ T cells in Stat3fl/flBatffl/flCd4Cre+ mice, which may serve as useful model to study γδ T cells in IBD-like colitis. Given that current therapies for IBD patients are only partially effective, the resulting data from this study and the establishment of this unique mouse model of γδ T cells in IBD-like colitis will potentially identify novel therapeutic targets to make a positive impact on the 6.8 million people living with IBD worldwide.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Candida auris (CA), an emerging multi-drug resistant fungal pathogen that predominately colonizes in the skin has recently been classified as an urgent threat by the US Centers for Disease Control and Prevention (CDC) Antibiotic Threats Report. CA asymptomatically colonizes the skin for prolonged periods and rapidly spreads between hospitalized patients and nursing home residents via nosocomial transmission resulting in outbreaks of systemic infections. CA gains access to the blood in patients requiring indwelling devices leading to invasive infections resulting in mortality ranging from 40 to 60 percent among CA infected patients. Majority of CA isolates exhibit resistance to all three classes of FDA-approved antifungal drugs including azoles, polyenes, and echinocandins posing a significant challenge to treat this fungal pathogen. Therefore, understanding the factors regulating CA skin colonization is important to control CA outbreaks and prevent invasive CA infections. Recently, we have uncovered that skin microbiota regulate the CA skin colonization. Based on our compelling data, we will define the microbiota and host mediated mechanism(s) that control CA skin colonization. In Aim 1, we will define how skin microbiota directly inhibit CA growth and skin colonization and In Aim 2, we will define the host-mediated mechanisms through which skin microbiota regulate CA skin colonization. Understanding the microbiota and host factors in the regulation of CA is expected to lead to a more complete understanding of the factors that control CA skin colonization, with the long-term aim of identifying new antifungal therapeutic strategies.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Alzheimer’s disease (AD) is the leading cause of dementia worldwide currently affecting over 50 million people. The pathophysiology of Alzheimer’s dementia is complex and multifactorial, however, decreases in cerebrovascular function have been linked to disease progression. Despite the role vascular health plays in the prognosis of AD, the ability to assess intracranial vascular integrity is limited. There is a critical need to assess vascular properties sensitive to microvascular function and arterial stiffness to understand why and how vascular health is a substantial risk factor for AD dementia. The right tool will be able to assess multiple cerebrovascular health metrics and monitor potential interventions targeting the vascular system in the treatment of AD dementia. This study aims to utilize a conventional resting-state functional MRI (rs-fMRI) scan to assess arterial stiffness and microvascular health through the development and implementation of specialized image processing techniques. These metrics will be applied to two large datasets available from the Human Connectome Project (HCP) – Aging and the Indiana Alzheimer’s Disease Research Center (IADRC). Using the HCP dataset, we will assess vascular changes in a typically aging sample (aims 1 & 2). With the IADRC dataset, we will assess the associations of cerebrovascular health with cognitive function across a spectrum of cognitive impairment (aim 3). The central hypothesis is that measures sensitive to arterial stiffness and microvascular function derived from specialized rs-fMRI image processing will significantly correlate with typical aging and cognitive impairment in the spectrum of AD pathology. The central hypothesis will be tested with the following aims: Aim 1: Assess the correlation of cerebral artery stiffness and age using rs-fMRI-derived arterial pulse propagation mapping. Aim 2: Evaluate rs-fMRI-derived cerebral transit time (CTT) in the HCP-aging dataset. Aim 3: Determine whether rs-fMRI-derived arterial stiffness and CTT in the AD-spectrum are significantly associated with cognitive impairment. The measures of arterial stiffness and microvascular function will provide greater insight into the influence of vascular health on AD dementia and may be used in the future to monitor intervention status. The entire pipeline with detailed demo code developed in this project will be openly shared to allow other researchers to extract these vascular metrics from standard rs-fMRI data and study other pathologies with known cerebrovascular involvement, resulting in a high clinical impact.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Heterogeneous noble metal catalysts are commonly used in organic synthesis for the hydrogenation of unsaturated organic functionality because of their fast reactivity and ease of purification. However, heterogeneous catalysts tend to be poorly selective in the presence of other reducible functionality and incapable of achieving stereoinduction due to the two-dimensional nature of the catalyst surface. Taking inspiration from the homogeneous literature on substrate- directed reactivity, the Li group recently demonstrated the first example of a heterogeneous hydroxyl-directed hydrogenation. Using a bimetallic alloy catalyst containing a noble metal and a base metal, we were able to catalyze highly diastereoselective hydrogenations of cyclic olefins by simultaneously adsorbing the hydroxyl directing group onto the base metal atom and the olefin onto the noble metal site. The overarching goal of this proposed research is to demonstrate that directed reactivity using bimetallic alloys is a general strategy to achieve chemo- and stereoselective hydrogenation of drug-like compounds. These selective hydrogenation reactions will be utilized to increase the sp3 content, three-dimensionality, and structural diversity of pharmaceutical candidates. We will develop new bimetallic nanoparticle compositions in order to extend the directed hydrogenation concept to systems that are not accessible using molecular catalysts, including arenes and heteroarenes, amine-directed reactions, and substrates where the directing group and reactive moiety are located remote from one another. We will also explore directing effects in dictating chemo- and regioselectivity in substrates where multiple reducible functional groups are simultaneously present. In parallel with the development of synthetic methods, we will conduct detailed nanomaterials characterization, in-situ surface spectroscopy, and kinetic studies in order to elucidate the surface ensemble required for high directivity. Together, this research program will provide new heterogeneous catalysts and methods for selective, late-stage transformations in biologically-active compounds.

NIH Research Projects · FY 2026 · 2023-08

The dynamics of covalent post-translational modifications (PTMs) on histones are a key mechanism in epigenetic regulation. Histone PTMs (also known as the “histone code”) are dynamically introduced and removed by “writer” and “eraser” enzymes, while the recognition of these PTM makers by “reader” proteins controls the activation and suppression of specific genes, motivating downstream epigenetic effects. Histone monoaminylation (i.e., H3 serotonylation and dopaminylation) is a newly identified epigenetic marker that plays an important role in regulating neuronal transcription, both during development and in the adult brain. Transglutaminase 2 (TGM2) has been proved to serve as the writer enzyme for this emerging histone PTM, which installs serotonin or dopamine onto the N-terminal glutamine residue of H3 (i.e., H3Q5) through transamidation. However, the eraser and reader for H3Q5 monoaminylation still remain elusive. In our recent study, we applied chemical biology approaches to understand the dynamic control of histone monoaminylation and unexpectedly discovered that the installation, removal, and replacement of this modification are all mediated by a single enzyme, TGM2. The biochemical mechanism of this novel regulation is attributed to the formation of a reactive thioester complex between TGM2 and H3 that can be attacked by nucleophiles (such as diverse monoamine metabolites). Based on this unique enzymology, we identified an unreported histone monoaminylation, H3Q5 histaminylation, and found that this new epigenetic marker promotes neural rhythmicity through epigenetic regulations. In this research program, we will develop a series of chemical probes that can orthogonally label and enrich different histone monoaminylations. Utilizing these probes, we will identify new types of monoaminylations (especially the ones caused by gut microbiome-derived monoamines) both in vitro and in vivo. Thereafter, we will demonstrate the pathophysiological roles of these epigenetic makers. We will also design and synthesize photocrosslinker- containing monoaminylated peptides as chemical baits to covalently capture possible readers that recognize and bind the target monoaminylations. The epigenetic functions of these identified readers will be further validated both in vitro and in vivo. Finally, we will employ the chemical probes developed in this study to characterize the non-histone targets (such as transcription factors) of monoaminylations and elucidate their potential roles in epigenetics and chromatin biology. Together, these findings will expand the categories of histone code and open a new door towards understanding the interplay between monoamine metabolism (from either host cells or gut microbiome) and cell fate regulation.

NIH Research Projects · FY 2026 · 2023-07

Neovascularization in the retina or choroid of the eye is a key feature of major causes of blindness such as neovascular age-related macular degeneration and proliferative diabetic retinopathy. Anti-vascular endothelial growth factor biologics have greatly aided treatment, but resistance to therapy, side effects, frequent intravitreal injections, and high cost remain significant limitations, creating a critical need for new therapy. Previous research revealed an appealing new target for the development of such therapies: the heme synthesis enzyme ferrochelatase (FECH), which, when inhibited or genetically modified, blocks neovascularization. The approved anti-fungal drug griseofulvin is naturally metabolized in vivo to form a FECH inhibitor. Griseofulvin thus blocks angiogenesis in vitro and in retinal and choroidal neovascularization animal models, offering promise for “repurposing” this old drug for ocular neovascularization treatment. However, griseofulvin has not been optimized for ocular use. For griseofulvin to be competitive with existing therapeutic modalities, it must be made available in a long-acting formulation for intravitreal use. Preliminary data reveal that griseofulvin can be formulated into polymeric implants and polymeric microparticles, which are amenable to sustained release over at least two months and effective against laser-induced choroidal neovascularization (L-CNV) weeks after application. Given this feasibility, the long-term goal is to provide a safe and affordable alternative to existing biologic agents by developing long-acting griseofulvin systems. The hypothesis is that long-term griseofulvin delivery can prevent ocular neovascularization. The hypothesis is based on prior research supporting FECH as an effective antiangiogenic target and griseofulvin as an indirect inhibitor of FECH. Polymeric implants and particles are well- received, long-term ocular drug delivery systems. With combined expertise in formulation, drug delivery, and neovascular eye disease, and preliminary results supporting controlled griseofulvin release, the team is poised to develop long-acting griseofulvin systems as new local therapies via three specific aims: 1. To optimize release kinetics of griseofulvin-encapsulated polymeric microparticles and implants. Poly(lactic-co-glycolic acid) microparticles and hot melt extruded polymeric implants will be developed to achieve 2-12 month delivery and characterized biophysically and in vitro. 2. To evaluate griseofulvin microparticles for antiangiogenic effects. Optimized microparticle formulations will be tested for long-term drug release in vivo, toxicity, efficacy, and target engagement in the L-CNV and Vldlr-/- subretinal neovascularization mouse models. 3. To evaluate griseofulvin- releasing polymeric implants for antiangiogenic effects. The optimized implant formulation will be tested for long- term drug release in vivo, toxicity, and efficacy in the DL-aminoadipic acid rabbit retinal neovascularization model. The core innovation of this strategy is the repurposing of griseofulvin for ocular neovascularization therapy by creation of sustained release formulations. If successful, these formulations would inhibit the progression of neovascularization with minimum inconvenience to the patients and cost to the healthcare system.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Epigenetic dysregulation is frequently observed in pediatric cancers, including neuroblastoma (NB), the most common extracranial solid tumor in pediatric patients. In my postdoctoral work, I identified that a cell state transition from an adrenergic to a mesenchymal epigenetic state is associated with the loss of GD2 expression and resistance to anti-GD2 therapy. Given the important role of anti-GD2 therapy in treating high-risk neuroblastoma patients, I designed a CRISPR-Cas9 screening platform to study epigenetic regulators of GD2 expression. I identified that individual knockout of several members of the PRC1.1/BCOR complex increases GD2 expression in GD2-low cell lines. AIM 1 will establish the relationship between the PRC1.1/BCOR complex and GD2 regulation by rigorously testing the necessity of the PRC1.1 complex to maintain low ST8SIA1 expression in mesenchymal cell lines. Mining genetic dependencies across 25 tumor lineages, I identified that the PRC1.1 complex is an enriched dependency in neuroblastoma independently of its ability to regulate GD2 expression. AIM 2 will validate that the gene PCGF1, the top enriched PRC1.1 subunit dependency in neuroblastoma, is a genetic dependency in multiple models of neuroblastoma. I will intersect chromatin and single-cell RNA-sequencing studies to determine the consequences of PCGF1 knockout on chromatin regulation and differentiation/cell state trajectories. No known small molecule inhibitors of PRC1.1 currently exist. The correlation of USP7 genetic dependency in the Dependency Map portal against all other gene dependencies revealed a strong correlation with PCGF1 dependency, suggesting a tractable pharmacologic approach to inhibiting PRC1.1. AIM 3 will establish USP7 inhibition as a mechanism to modulate PRC1.1 activity. These specific aims will test the capacity of highly potent and selective USP7 inhibitor to selectively upregulate GD2 expression and reduce neuroblastoma viability in vitro and in vivo. I anticipate that these findings will directly link PRC1.1 to epigenetic state and differentiation in neuroblastoma. Moreover, it will credential USP7 inhibition as a combinatorial therapy to restore the response to anti-GD2 therapy and directly target neuroblastoma cells. To complete the studies in this proposal, I will apply my strong expertise in epigenetics and pharmacology. To fill in critical gaps in knowledge and expand my scientific training, I’ve assembled a training plan that includes advisory committee members that are experts in immuno-oncology, single-cell sequencing, and USP7 chemistry. This proposal lays a strong framework for my long-term goal of establishing a lab that focuses on targeting epigenetic plasticity/heterogeneity as an intervention to overcome therapeutic resistance.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Blood hemoglobin (Hgb) testing is a common clinical laboratory test during routine patient care and screening. In particular, blood Hgb tests are essential for the diagnosis and management of anemia. Globally, over 40% of pregnant women are anemic, adversely affecting maternal and fetal health outcomes through increased morbidity and mortality. A range of treatments for anemia are well-established and readily available even in low- and middle-income countries. In these settings, the main challenge is that anemia is not detected or detected too late. For pregnant women in resource-limited settings who require several Hgb tests during all trimesters, conventional invasive blood Hgb tests are not only painful and iatrogenic, but are also expensive and often inaccessible. Existing noninvasive devices and smartphone-based technologies for measuring blood Hgb levels often rely on costly specialized equipment and complex smartphone attachments, thus hampering practical translation from research to clinical practice in resource-limited settings. Based on the preliminary results generated by our transdisciplinary team, we hypothesize that blood Hgb levels can be accurately and precisely predicted from a red-green-blue (RGB) image of the inner eyelid (palpebral conjunctiva) acquired using a smartphone camera with no additional attachments, and that this mobile health (mHealth) application can be fully integrated with an existing electronic health record (EHR) system in low-resource settings. Specifically, an informed learning approach will enable us to incorporate a physical or biological understanding into the learning algorithms to overcome the limitations of purely data-driven machine learning. Our team, consisting of experts in optical spectroscopy and machine learning, biomedical informatics and implementation science, and maternal and public health, proposes three aims to achieve the project goals. In Aim 1, we will develop a robust, simple, frontend data acquisition method for various mHealth and digital health settings. A tissue-specific color gamut design and true color recovery will provide the first-of-its-kind systematic methodology to realize color accuracy that will be highly sensitive to blood Hgb. In Aim 2, we will perfect the core mHealth computational algorithm using clinical data of black African pregnant women. Sub-algorithms of automated inner eyelid demarcation, advanced spectral learning, and blood Hgb content computation will enable fully automated, highly accurate, and precise blood Hgb estimations. Tissue optics-informed spectral learning will capture strong nonlinearity between RGB values and spectral intensity directly in the spectral domain. In Aim 3, we will integrate mHealth blood Hgb technology with a widely used EHR and evaluate the backend performance. The proposed connected mHealth technology will demonstrate the possibility of offering mobility, simplicity, and affordability for rapid and scalable adaptation, maximizing the currently available resources in resource-limited settings. Our work can also provide reciprocal innovation to offer advanced mHealth and digital health technologies combined with telemedicine in rural and at-home settings in the US.

NIH Research Projects · FY 2026 · 2023-04

Radiotherapy is an indispensable part of the standard care for glioblastoma (GBM) patients; however, despite initial responses to radiotherapy, GBMs invariably recur. A proposed strategy for improving GBM radiotherapy involves combining both radiotherapy and therapy targeting tumor-associated macrophages (TAMs). However, lack of an established mechanism by which TAM-targeted therapy improves GBM radiotherapy has posed a barrier to clinical translation. Thus, there is an urgent need to establish mechanisms by which TAM-targeted therapy alters radiotherapy. The main objective of this project is to determine the cytotoxic mechanisms and anti-tumor efficacy of integrating TAM targeting within clinically relevant radiotherapy regimens. For these studies, therapeutic targeting of TAMs will be accomplished by repurposing the FDA-approved agent ferumoxytol. The main hypothesis is that ferumoxytol will reduce immunosuppressive, tumor-promoting TAMs. This reduction in TAMs is expected to increase glioma cell sensitivity to radiotherapy by both disrupting TAM-glioma cell heterotypic survival signaling and increasing radiation-induced anti-tumor immune responses. This hypothesis will be evaluated using radiotherapy regimens that are similar to clinical standards of care for two types of patients: those with newly diagnosed GBM (Aim 1); and those with recurrent GBM (Aim 2). This will include completion of the following aims: Aim 1) determine cytotoxic mechanisms and efficacy of combining ferumoxytol with conventionally fractionated radiotherapy; and Aim 2) evaluate ferumoxytol’s ability to augment hypofractionated radiotherapy. Aim 1 will evaluate the combination of ferumoxytol with radiotherapy in both in vitro coculture models and syngeneic rodent models. Aim 2 will evaluate the combination of ferumoxytol and hypofractionated radiotherapy in a translationally-relevant canine companion study. For both aims, the delivery and retention of ferumoxytol within tumor regions will be verified non-invasively using magnetic resonance imaging (MRI). The investigators believe the proposed research is innovative because it repurposes an established glioma imaging agent (ferumoxytol), for theragnostic TAM targeting. If ferumoxytol does not prove as effective as expected for enhancing radiotherapy, these studies will shift focus to other promising TAM-targeted therapeutics being developed by the investigative team. Upon completion of the proposed studies, this project will have established the therapeutic efficacy and cytotoxic mechanisms of combining TAM-targeted therapy with radiotherapy. This contribution is expected to be significant for two reasons: it will provide knowledge regarding the role of TAMs in modulating glioma cell resistance to radiotherapy; and it will lead to development of more effective radiotherapeutic strategies. Given the prevalence of radiotherapy for treating cancer, development of this therapeutic approach has the potential to improve outcomes for many patients.

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de doctorat en recherche; Domaine: Structures abstraites; Objet: Modélisation et simulation; Objet: Techniques de l'espace; Application: Sciences et technologies; Application: Fondements et avancement des connaissances; Mots-clés: PHYSIQUE, GENIE ASTRONAUTIQUE, SYSTEMES DYNAMIQUES, ASTRODYNAMIQUE, CONCEPTION DE TRAJECTOIRES SPATIALES, PROBLEME A PLUSIEURS CORPS

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT There are approximately four million referrals to child protective services (CPS) in the United States each year. Children and youth who receive adequate support often experience fewer negative physical, mental, and social health outcomes and are less likely to perpetrate violence in the future than their peers who received no or inadequate support. Younger children tend to exhibit signs and symptoms that help others to identify their need for intervention. However, young people are unlikely to discuss and report their experiences through traditional, face-to-face interactions. Technology-facilitated communication (e.g., text, chat, social media) is the preferred communication method for many young people. The candidate’s prior work demonstrated that many young people share their child maltreatment experiences through social media and other technology-facilitated platforms. Thus, technology-facilitated communication may be leveraged to support young people through their help-seeking experience and connect them with appropriate resources. The objectives of this K01 are twofold: 1) This K01 will support the candidate to gain additional training required for an independent, interdisciplinary research career developing and evaluating technology-based interventions that support young people while seeking maltreatment-related resources and information. Working with a team of experts in the fields of developmental psychology, child and adolescent health, bioethics (including online research and research with vulnerable populations), computational analytics, and digital health communication, the candidate will gain 1a) knowledge and expertise in research ethics related to technology and vulnerable populations, 1b) skills in computational data analytics and visualization, and 1c) experience in developing and implementing health communication interventions. 2) The research conducted through this K01 will build the empirical foundations for future interventions by 2a) identifying the language used by young people to discuss their maltreatment experiences (Aim 1), 2b) evaluating the feasibility of using technology to reach young people seeking maltreatment-related information and support (Aim 2), and 2c) developing and piloting technology-facilitated interventions that connect young people to appropriate resources (Aim 3). After completing the training and research activities proposed in this K01, the candidate will be uniquely prepared to launch a career as an NIH- funded researcher investigating novel approaches to preventing the negative health consequences of child maltreatment.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Opportunistic fungal infection of immune-compromised individuals is an escalating world health problem. Recent lethal outbreaks of multi drug-resistant Candida auris in hospitals and the rise of drug resistance in normally benign commensal fungi like C. glabrata highlight the severity of the problem. Current treatment options for fungal infections are limited to a few antifungal drug classes that are becoming increasingly ineffective. There is a pressing need for new molecular targets for antifungal development to deal with drug- resistant pathogens. This project will characterize a newly identified C. albicans virulence and drug resistance factor, the Cdc14 protein phosphatase. Our recent work has uncovered novel roles for C. albicans Cdc14 in regulating cell wall integrity, septation, echinocandin sensitivity, and hyphal development, all processes tied to virulence. Importantly, even modest reduction in Cdc14 activity level severely compromises virulence in a mouse model of invasive candidiasis. In contrast, Cdc14 is dispensable for normal development, growth, and cell division in animals. Cdc14 is highly conserved in fungi and its unique and strict active site specificity implies that development of potent and highly selective inhibitors should be achievable, something that has been challenging with other protein phosphatases. Our overall objective is to characterize the mechanisms by which Cdc14 regulates virulence-associated biological processes in C. albicans. In Aim 1 we will characterize Cdc14 regulation of cell wall integrity and septation. In Aim 2 we will characterize Cdc14 regulation of hyphal initiation and maintenance. In Aim 3 we will characterize the mechanisms by which Cdc14 itself is regulated by cell wall stress and hypha-inducing signals. In Aims 1 and 2 we will employ unbiased omics approaches to identify the relevant substrates of Cdc14 and the transcriptional circuits under Cdc14 control. In Aim 1 we will directly characterize the cell wall defects arising from Cdc14-deficiency. We will also test specific models for Cdc14 function in promoting cell wall integrity and hyphal initiation in Aims 1 and 2, respectively. In Aim 3 we will focus on phosphoregulation of the disordered Cdc14 C-terminal tail, which is a hub for integration of regulatory signals in model fungi. We will use quantitative phosphoproteomics to understand the dynamic phosphorylation of C. albicans Cdc14 during cell wall stress, cytokinesis/septation, and initiation of hyphal differentiation. All three aims will conclude with structure-function analyses using biochemical, cell biological, and waxworm and mouse infection assays to characterize the physiological significance of Cdc14 function and phosphoregulation, including the importance for pathogenesis. Collectively, the results will define the molecular mechanisms by which Cdc14 promotes several virulence-related biological processes that will be useful in assessing its future potential as an antifungal target. The identification of Cdc14 substrates and effectors may provide additional candidate antifungal targets. The high conservation of Cdc14 structure, activity, and specificity across the fungal kingdom implies the results will be relevant to many other fungal pathogens.

NIH Research Projects · FY 2026 · 2023-02

Changes in metabolism in the aging eye can affect its epigenome because several metabolic intermediates also act as donor molecules for deposition of epigenetic marks such as histone and DNA methylation. During aging, there are changes in the metabolic pathways that produce the donor molecule required for histone and DNA methylation, S-adenosylmethionine (SAM), from the amino acid methionine. Methionine metabolism is strongly linked to aging across multiple species because restricting methionine intake extends lifespan, and is thought to be responsible for the lifespan extension caused by caloric restriction. In the aging Drosophila eye, we observe changes in methionine metabolism including an increase in levels of S-adenosylhomocysteine (SAH), which inhibits the activity of methyltransferases. This age-associated increase in SAH correlates with decreased levels of histone methylation marks across the entire genome in photoreceptors. Moreover, we show that loss of the methyltransferases that deposit one of these marks in photoreceptors leads to premature retinal degeneration. We propose that the decreased histone methylation in aging photoreceptors contributes to the age-related changes that we observe in gene expression. Specifically, we have identified changes in rhythmic expression of more than a third of active genes in aging photoreceptors together with altered transcription factor binding activity of the circadian master regulators Clock and Cycle. The circadian clock is highly conserved from flies to humans, and maintains biological rhythms by controlling gene expression programs through a series of transcription- translation feedback loops. When we disrupt the circadian clock in photoreceptors, we observe substantial retinal degeneration accompanied by global changes in chromatin accessibility and misregulation of more than a quarter of active genes. Loss of circadian regulators in the mouse eye causes age-dependent retinal degeneration, suggesting that the circadian clock has a conserved role in protecting the aging eye. Based on our preliminary data, we hypothesize that increasing oxidative stress in the aging eye inhibits activity of the sole enzyme that breaks down SAH on chromatin at actively expressed genes. We further propose that the local increases in SAH levels at expressed genes inhibit the activity of histone methyltransferases, leading to changes in the rhythmic expression of genes in the aging eye. Together, these studies provide a framework in which to understand how the normal changes that occur in the aging eye can disrupt its metabolism, leading to changes in the epigenome that disrupt normal patterns of gene expression and increase the risk of ocular disease. Drosophila provides an ideal model for these studies because it shares a similar circadian clock and epigenetic mechanisms with humans, but ages much more rapidly allowing us to examine mechanisms in the context of normal aging in specific cell types in the eye.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY While significant evidence has demonstrated that obesity increases the risk of metastasis, the molecular mechanisms by which obesity contributes to the metastatic progression of breast cancer are unclear. Furthermore, recent research in cancer development and progression has highlighted the role of hypoxia and dysregulated lipid metabolism. Research from our team and others demonstrate that lipid accumulation, which is associated with reduced patient outcomes, is greater in metastases compared to primary tumors. Furthermore, hypoxia, which is increased in obesity in the primary tumor, leads to sustained increase in the expression of specific genes after reoxygenation, a hypoxic memory. Our results demonstrate that hypoxic memory results in the expression of fatty acid synthase (FASN), which is the rate-limiting step in fatty acid synthesis, and pyruvate carboxylase (PC), which we have shown provides oxidative stress protection. In addition, the inflammatory cytokine interleukin-6 (IL-6), which is elevated in obesity, enhances the expression of CPT1A, the rate-limiting step in fatty acid oxidation (FAO) to supply energy. Our preliminary data show that the expression of these three proteins and their functional consequences of increased fatty acid synthesis and FAO are elevated in metastases compared to primary tumors. Thus, our preliminary results suggest that hypoxia may set the stage for dysfunctional lipid metabolism, where increased lipid synthesis and utilization occur concurrently with a balance towards lipid accumulation. However, the impact of dysfunctional lipid metabolism in obesity-driven metastasis is unknown despite the supporting evidence that hypoxia and IL-6 are enhanced in obesity. In the proposed studies, the research team will utilize multiple mouse models of obesity and metastatic breast cancer to evaluate the mechanistic basis by which hypoxic memory and IL-6 interact to stimulate obesity-driven breast cancer metastasis. They will test the hypothesis that obesity-associated increases in hypoxic memory and proinflammatory IL-6 signaling work in tandem to increase FA accumulation (FASN), FAO (CPT1), and cell survival (PC) to enhance metastases. These hypotheses will be tested through the completion of the following two aims: 1) determine the impact of hypoxic memory on lipid accumulation in obesity-driven metastasis, and 2) establish the interaction of hypoxic memory with chronic inflammation in obesity-driven metastasis. These studies will provide foundational evidence for developing targeted strategies to mitigate obesity-driven metastatic breast cancer.

NIH Research Projects · FY 2026 · 2022-11

PROJECT SUMMARY The German cockroach, Blattella germanica, is the most common pestiferous cockroach species in human environments. B. germanica contributes to the transmission of bacteria that cause enteric (diarrheal) disease, including Salmonella enterica serovar Typhimurium, but the mechanisms of transmission are not well understood. Enteric bacterial pathogen transmission by cockroaches has previously been described as mechanical in nature. Mechanical transmission is a passive, non-replicative transfer of bacteria from one location or host to another. This mechanism is limited in impact relative to active biological transmission. However, recent data from our laboratory indicate that transmission of S. Typhimurium by German cockroaches is markedly more complex than simple mechanical transmission and instead resembles biological transmission by other insects that intake bacteria from infected hosts and are subsequently colonized, enabling active and prolonged shedding and transmission. In particular, we have observed that following ingestion, S. Typhimurium undergoes a lifestyle change and multiple replication events in the digestive tract of the German cockroach. Furthermore, we have identified several S. Typhimurium genes that are necessary for bacterial colonization and shedding from the gut of B. germanica, evidencing an active role of the bacteria. The central objective of the proposed research project is to gain a detailed understanding of the mechanisms of biological vector-borne transmission of S. Typhimurium by the German cockroach. Three independent specific aims are proposed. First, we will elucidate the fine spatiotemporal details of wild-type S. Typhimurium colonization and shedding in nymph and adult cockroaches. Second, leveraging an unparalleled array of mutant S. Typhimurium strains, we will identify specific bacterial genes and functions that are necessary for colonization of cockroaches and subsequent transmission. Third, we will determine the effects of two key host factors, namely antimicrobial effectors and the gut microbiota, on S. Typhimurium colonization and shedding. Together, the proposed studies will establish a picture of how bacterial and host factors interact to shape biological transmission of S. Typhimurium by the German cockroach, providing fundamental insight into the dynamics of a unique, poorly studied vector-pathogen system with a global distribution and public health impact.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Scaffolded stem cell transplantation has the potential to provide structured chemical and mechanical cues to guide cell growth into functional tissues for regenerative medicine. However, significant challenges have limited the clinical success of this approach. Cells implanted in large scaffolds often have limited viability due to hypoxic conditions in the host, while cells injected individually or in small clusters often suffer from poor retention at the site of injury. Hydrogels are commonly utilized as stem cell scaffold materials because they confer substantial flexibility in terms of chemical composition and ligand integration. However, hydrogels are also typically amorphous, limiting control over ligand presentation (important for adhesion and signaling), and lacking structural cues such as fibers that are present in biological ECM, which impacts mechanical strength. We have recently demonstrated that it is possible to generate stable, 1-nm-resolution functional patterns on amorphous polyacrylamide and polydimethylsiloxane surfaces, using sub-nm-thick films of highly ordered polydiacetylenes (PDAs) that are preassembled and covalently transferred to the hydrogel surface. Our approach potentially addresses both chemical and mechanical challenges associated with hydrogel stem cell scaffolds, enabling generation of cell-instructive hydrogel tapes that can be shaped to create 3D scaffolds. However, to be useful in clinical settings, this strategy will need to be validated with: (1) commonly used hydrogel stem cell scaffold materials, (2) hydrogel moduli matching the range commonly associated with tissues, and (3) films thin enough for adequate perfusion to prevent hypoxia and enable normal secretome interactions. Here, we develop a platform technology based on cell-instructive hydrogel tapes, benchmarking their chemical and mechanical properties, and their impacts on human mesenchymal stem cells (hMSCs). In Aim 1, we evaluate the hypothesis that PDA surface functionalization can improve chemical control over surfaces of hydrogels common in regenerative medicine, orienting and spatially clustering ligand presentation, to modify stem cell growth in a predictable fashion. We test this by culturing hMSCs on surfaces designed to maintain stemness or to induce specified differentiation behavior (angiogenesis, adipogenesis, chondrogenesis), benchmarking against common stochastic hydrogel modification strategies. In Aim 2, we evaluate the hypothesis that our PDA surface-functionalization approach can improve the mechanical and handling properties of thin, soft hydrogel films, enabling creation of cell-instructive hydrogel tapes. We generate and test impacts of paired tapes as cell sandwich scaffolds and 3D constructs that provide structured chemical surfaces and mechanical environments, while maximizing perfusion to and from cells. Overall, this proposal develops a modular surface functionalization strategy that can be easily integrated with many existing hydrogels for tissue scaffolds, providing structured ligand presentation and mechanical strength aimed at improving utility in clinical cell transplantation therapies.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Antibiotic resistance of bacterial pathogens is one of the greatest public health challenges of our time. It causes difficult-to-treat infections and jeopardizes modern healthcare advancements. As the emergence of bacterial resistance is outpacing the development of new antibiotics, we must find cost-effective, innovative approaches to discover new antibacterial therapeutics complementary to small-molecule antibiotics. Antimicrobial peptides (AMPs), as a new class of antibacterial agents, represent one of the most promising solutions to fill this void, since they generally undergo faster development, display rapid onsets of killing, and most importantly show lower risks of induced resistance, compared to small-molecule antibiotics. Yet, very few analogs or modified derivatives of natural AMPs have been approved in practice, and most of the failure is caused by systemic or local toxicity associated with broad-spectrum antibacterial activity. Toward a long-term goal to discover effective, selective AMPs as therapeutics to target a narrow spectrum of specific antibiotic-resistant pathogens, our objective is to develop the new capacity needed for such discovery, by integrating innovative approaches and applications of machine learning, multiscale modeling, peptide synthesis, and microbiology. We have developed the first generative adversarial network model (AMP-GAN) to produce AMP candidates with diverse sequences and structures, as well as accurate multiscale models and methods to study the mechanisms of AMP aggregation and target interactions. It is our central hypothesis that AMP selectivity may be achieved via controlling their sequence, structure, interaction, aggregation, and co-aggregation. In pursuit of three specific aims to establish a novel methodology toward discovery of narrow-spectrum AMPs, we will (i) generate selective AMP sequences with predictable activity and pathogen targets, (ii) identify AMPs to target characteristic biomolecules in pathogens, and (iii) modulate AMP aggregation to tune cell selectivity or to achieve synergy. We will advance our computational techniques like AMP-GAN and top-down simulations in conjugation with chemical characterizations (for structure and dynamics) and cellular assays (for activity and toxicity). We anticipate gaining a fundamental understanding of how to design narrow-spectrum AMPs, as well as how to combine new computational and experimental tools to achieve desired AMP selectivity. Overall, this contribution can be significant since it will establish new avenues for precision AMP design and bring more AMPs closer to the clinic by overcoming their known pitfalls. The resulting knowledge will be widely shared in the scientific community for AMP research and development. Our concepts and approaches are innovative, as they shift the current paradigm of broad-spectrum AMP design towards higher accuracy, diversity, and target selectivity through precision AMP design. Collectively, given the increasing need for treatment options against antibiotic-resistant infections, the methodology and tools from this proposed research will enable the discovery of new therapeutics for challenging infectious diseases.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT With the spread of COVID-19, public health precautions have required physical distancing and a variety of shelter-in-place orders, causing rapid and substantial shifts in all aspects of family and community life. Data from COVID-19, supported by evidence from past national emergencies, suggest caregivers are at clear risk for secondary health effects because of the current pandemic. COVID-19 has been described as a “perfect vector for a mental health epidemic” due to the accumulated effects of pandemic-related fear, social isolation, post-traumatic stress, and mental health treatment barriers. These changes to daily life have been especially challenging for families of children with neurogenetic conditions (NGC), who experience high levels of baseline stress, are particularly vulnerable to reductions in targeted therapeutic services, and rely on medical and educational services that have been disrupted by COVID-19 related closures. Specifically, caregivers themselves are vulnerable to stress-related mental and physical health challenges because of the pandemic; they rarely receive treatment for their own mental health needs, and any treatments they do receive are typically disconnected from their child's care plan and are delivered by practitioners with little-to-no expertise in the needs of NGC families. These health care gaps are amplified among Black and other minority families. The proposed study will address the needs of caregivers of children with NGC by examining the feasibility, efficacy, and impact of a novel network for delivering personalized triage and digital treatment. Specifically, we propose to scale up and integrate a series of brief, evidence based digital health interventions to support caregiver mental health, parenting self-efficacy, and stress. Supervised graduate student trainees will implement rigorous, cost-effective, evidence-based interventions via telehealth. A key innovation of this work is that we will develop a personalized health routing algorithm that matches participants with relevant treatments using both clinical inputs and ecological momentary assessment (EMA) data, brief questionnaires “pinged” to caregiver phones via a smartphone app. A second innovation is that we will test the efficacy of peer-to-peer coaching in enhancing treatment uptake and outcomes. Coaching will be delivered by fellow NGC caregivers using an evidence-based motivational interviewing protocol. By the end of the award period, we will have generated a novel, scalable, and cost-effective solution for rapidly meeting acute needs for NGC caregivers through personalized, digital delivery of evidence-based treatments. This model can be rapidly scaled for other high-risk populations during COVID-19 (e.g. first responders, teachers, frontline workers) and future public health crises. Given substantial unmet needs existed among NGC families even prior to COVID- 19, this protocol has potential to fundamentally shift the status-quo for how treatment is selected and accessed in NGC caregivers and other underserved groups, including beyond the COVID-19 pandemic.

NIH Research Projects · FY 2023 · 2022-09

PROJECT SUMMARY High-energy trauma to an articular joint delivers a mechanical overload to cartilage tissue and causes an injury response in chondrocytes that frequently leads to post-traumatic osteoarthritis (PTOA). Because cartilage has limited intrinsic repair capabilities, there is an unmet clinical need for new therapies to treat cartilage injury and inhibit progression of PTOA. The mechanosensitive signaling pathways that mediate the injury response of chondrocytes to mechanical overloads are not well understood. Filling these gaps in knowledge may provide new therapeutic targets following joint injury that prevent or delay the development of PTOA. Our preliminary data identify Sirtuin1 (SIRT1), an NAD+-dependent protein deacetylase, as a newly- discovered mechanosensitive signaling molecule in chondrocytes’ response to injurious overload. SIRT1 activity decreased in bovine cartilage explants within 5 minutes of a sublethal impact overload, and remained suppressed for at least 24 hours. Preliminary experimental results also suggest the likely pathway that regulates SIRT1 deactivation. Importantly, pharmacological activation of SIRT1 prevented the acute injury response in the cartilage explants. The first objective of this study is to define upstream signaling pathways that regulate SIRT1 deactivation in the injurious mechanoresponse of chondrocytes. This will be accomplished in Aim 1 using pharmacological inhibitors and a CRISPR/SaCas9 strategy. Additionally, the major mechanism for SIRT1 to regulate cellular processes is to deacetylate proteins. Therefore, a second objective is to analyze the acetylome to determine the downstream substrates of SIRT1 in mechanically loaded chondrocytes, and to clarify the role of these deacetylation targets in the chondrocyte injury response and/or chondrocyte behavior. This objective will be met in Aim 2 with a proteomics approach to identify deacetylation substrates with mechanical overload. The role of at least one of these targets in the injury response to mechanical overload and/or cartilage health will be evaluated. The proposed study breaks new ground, as it investigates the mechanically induced enzymatic deactivation of SIRT1, which had not previously been identified as mechanosensitive in chondrocytes, in the impact injury response in cartilage. As sirtuins were initially recognized as pivotal regulators of aging and longevity, successful completion of the proposed work may provide a molecular link between injury-induced and age-related osteoarthritis, transforming our understanding of the disease. Furthermore, understanding the signaling pathways that are upstream and downstream of SIRT1 enzyme deactivation will identify new molecular events in the injury response of chondrocytes and may reveal previously undiscovered regulators of cartilage health. These results are expected to form the basis for new approaches to treat cartilage injury and novel therapeutic targets to induce articular cartilage repair.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Developmental exposure to heavy metals, such as lead (Pb), causes systematic damage to the central nervous system and impairs many neurological targets. Some of these biological perturbations, such as altered synaptic plasticity and endosome trafficking, are shared with Alzheimer's Disease (AD). Epigenetic mechanisms, given potency and latency in gene regulation, offer a plausible route to relay impacts from early-life environmental exposure events to AD. The exact molecular mechanism, however, remains elusive. The goal of this proposal is to define the epigenetic mechanism contributing to altered synaptic plasticity arising from developmental Pb exposure addressing the contributions of gene-by-environment (GxE) interactions in accelerating the progression of AD. Our preliminary studies and prior literature suggest persistent alterations in synaptic plasticity, primarily arising from changes in glutamate receptors, including NMDAR and AMPAR. Alterations in endosomal trafficking are also heavily implied. We formulated our central hypothesis that developmental Pb exposure alters the transcription of glutamate receptors via epigenetic regulation affecting synaptic plasticity with the effects exacerbated when coupled with the AD genetic risk factor, SORL1. This GxE interaction compromises endosomal trafficking and glutamate receptor recycling, which eventually leads to the onset of an AD-like phenotype manifested by protein aggregation markers. We adopted a multiplex model including cortical neurons derived from human induced pluripotent stem cells (hiPSCs) and a zebrafish animal model with and without a known late-onset AD (LOAD) risk factor (SORL1). We designed our experiments to dissect contributions from environmental (E), genetic (G), and GxE driven events in altering synaptic plasticity and the manifestation of AD- like phenotypes. We will test our hypothesis in three aims. Aim 1 will elucidate the impact of developmental Pb exposure and SORL1 effects on neuron susceptibility of protein aggregates. Aim 2 will reveal the molecular origin conferring developmental Pb neurotoxicity to an AD-like phenotype. Aim 3 will define subcellular alterations in the post-synapse associated with an AD-like phenotype. Collectively, we will curate time-dependent information about molecular changes in the transcriptome and epigenome, along with alterations in ultrastructure of post-synaptic spine. We will use the aggregated information to infer causative relations among different events by assuming early events are likely to drive late ones. We expect that GxE interactions arising from developmental Pb exposure and SORL1 mutation to induce a phenotype closely resembling AD, followed by SORL1 mutation only, Pb exposure in wild type, and untreated wild type. Furthermore, we will reveal novel targets mediating the latent effects of developmental Pb exposure on neurodegeneration risks via mining of our dataset and verification of the efficacy of underlying epigenetic profiles of glutamate receptors in accelerating AD onset and progression risks. The knowledge generated will enlighten the molecular mechanism of Pb neurotoxcity and connections of early life Pb exposure to AD progression addressing goals of PAR-22-048.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Activity from steel, petroleum, and chemical industries has resulted in extensive metal and organic compound contamination in northern Lake County, Indiana. Residents have consistently voiced the need for more community input and unbiased data on the extent and the health impacts of their chemical exposures. Many of these contaminants, individually, are recognized as neurological toxicants; however, scientific understanding of the cumulative impact of exposure to multiple contaminants remains unclear. Therefore, our goal is to conduct a community-engaged assessment of the patterns of exposure to metals and volatile organic compounds (VOCs) as well as their potential associations with selected health outcomes, particularly neurological health. We will accomplish this through completion of a cross-sectional study of 300 adults and 100 children from northern Lake County, Indiana. Community engagement will be achieved through a) engagement with a Community Advisory Board (CAB) to provide advice on all aspects of the project; b) employment of residents to serve as Community Health Workers (CHWs) to work closely with individual residents to share information about the project, recruit participants, and conduct study visits; c) empowerment of participants by involving them in collecting their own environmental and biological samples through a specialized sampling kit; and d) community outreach. In Aim 1, we will determine a) toxic metal concentrations in house dust, soils, tap water, hair and toenail samples as well as b) benzene, toluene, ethylbenzene, xylene, polycyclic aromatic hydrocarbons and other common VOCs in ground-level air samples and silicone wristbands. Fine particulate matter (PM2.5) in ambient air samples will also be determined. CHWs will show participants how to collect samples from their home using the sampling kit. The kit components have been validated; samples will be analyzed by scientific staff using semi-automated X- ray fluorimeters (XRF), inductively coupled plasma mass spectrometry (ICP-MS) and gas chromatograph mass spectrometers (GC-MS). In Aim 2, we will assess the correlation of mixed chemical exposure with self-reported general health status, cognitive and emotional function, and epigenetic profiles. Trained study staff will be trained to administer the cognitive battery from the NIH Toolbox via videoconference. Epigenetic changes include altered methylation patterns in genes known to be related to neurologic disease. In Aim 3, we will evaluate the effectiveness of our community engagement strategies. Specifically, we will explore whether these strategies are effective at promoting participation in project activities and increasing the knowledge of environmental and health issues. At the conclusion of this community engaged research, we will have completed a high-quality assessment of the extent and characteristics of exposure to metals and VOCs in northern Lake County; determined correlations of these exposures with selected health outcomes; and determined which community engagement methods were most effective in this community.

NIH Research Projects · FY 2025 · 2022-09

Project Summary / Abstract Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide, accounting for approximately half to two-thirds of all cases of dementia. It is associated with cognitive impairments and an increase in β-amyloid plaque (Aβ) deposits. Moreover, alcohol use disorder (AUD) has been associated with neurodegeneration and cognitive impairments, but the role of alcohol dependence on AD has been somewhat controversial. Recent evidence suggests a likely role for alcohol use as a risk factor in onset and severity of Alzheimer's disease, however overall reports have been mixed. Given the pervasive use of alcohol, it is critical to understand the potential impact of alcohol drinking on AD. We will examine the impact of a history of alcohol dependence on the onset and severity of behavioral and neuropathological symptoms associated with AD. We will further examine how alcohol dependence influences neural network function in AD. We will assess whether or not treatment with Memantine can reverse the behavioral and neuropathological symptoms. We hypothesize that a history of alcohol dependence will result in an earlier onset of cognitive impairment in AD mice as well as a greater presence of Aβ deposits and that these deficits will be rescued by chronic treatment with Memantine. We hypothesize that a history of alcohol dependence in AD mice will alter network connectivity (decrease modularity and change hub regions) and that these effects will be alleviated by Memantine treatment. Alterations of functional network connectivity that are caused by alcohol dependence will be assessed in a mouse model of AD. These changes will be compared with the brain-wide spread of Aβ deposits, which will indicate the way in which AD affects brain-wide function, potentially beyond brain areas with signs of neuropathology. Furthermore, the role of alcohol dependence in AD (e.g., changes in network connectivity and behavioral and neuropathological symptoms) will be examined using a relevant translational preclinical model of AUD. Cognitive function, anxiety-like behavior, and irritability-like behavior will be assessed in AD and wildtype alcohol drinking and alcohol naive mice. The number and location of Aβ deposits will be assessed AD mice. Differences in network connectivity in AD and wildtype mice will be examined using hierarchical clustering and graph theory. If alcohol dependence is found to impact the onset of neurological and behavioral symptoms of AD, then this will be a highly significant finding that will have a broad impact on preclinical and clinical research. These potential findings may also directly influence public guidelines for alcohol consumption, especially in those with a familial risk for AD.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The notion that the positions of chromosomal loci within the interphase nucleus are not static, but move, is not new. However, the mechanistic role of the chromatin motion is usually underestimated. In fact, the nanoscale motion of chromatin may modulate the interaction of DNA with regulatory molecules, including chromatin effectors, transcription factors and non-coding RNAs, thus impacting the global patterns of gene expression. Unfortunately, experimental evidence supporting an essential role of chromatin motion in these activities is sparse. Thus, it is unclear if changes in chromatin dynamics facilitate these biological processes or are simply consequences. The goal of this project is to quantify the chromatin motion throughout the mammalian nucleus and to explore its mechanistic role in genome functions. To this end, the proposed research combines multidisciplinary approaches to concentrate on the link of chromatin motion with epigenetic regulation, DNA damage, and transcription, with the ultimate goal to understand the causality from epigenetic modification to phenotypic gene expression. The proposed projects are listed as below, Project 1 Map the chromatin motion in 3D with nanometer resolution. We will heavily incorporate the data science approach into our innovative imaging system to optimize the toolbox of measuring the chromatin. The toolbox will include the data-driven 3D imaging system and data science supported image informatics software. Project 2 Investigate the link between chromatin motion and epigenetic modification and transcription. We will explore the reciprocal interactions between chromatin motion and epigenetic modifications as well as transcription, respectively. These experimental investigations can feed proper mathematical models, therefore to formalize the reaction network in chromatin remodeling and gene expression. Results generated from these projects have great potential to reveal fundamental links between chromatin motion and gene expression, which will transform our understanding of disease mechanism and facilitate the development of therapeutic intervention.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for the ongoing human pandemic (COVID-19) that has been classed as a Public Health Emergency of International Concern by the World Health Organization (WHO). There is an urgent demand for SARS-CoV-2 research to facilitate the development of therapeutics, understand viral replication and pathogenesis, and determine how the virus spreads from cell-to-cell as well as patient to patient. Coronaviruses such as SARS and MERS are among the most dangerous pathogens on Earth, with high fatality rates and lack of viable therapeutics or vaccines. They are classified as category C pathogens by the NIH due to their ease of production and dissemination with the potential of high morbidity and mortality. Detailed mechanistic studies on the dynamics of SARS-CoV-2 replication and viral shedding (i.e., budding) may inform identification of new drug targets in the viral life cycle and enrich our understanding of how this zoonotic pathogen utilizes host cell lipids to build the viral lipid envelope. The Stahelin and Voth laboratories, building on collaborations with each other and specific expertise in biochemistry, biophysics and computational studies of virus assembly, will use experimental in vitro and cellular studies integrated with computational analysis to investigate the central hypothesis in this grant: that selective lipid-protein interactions drive the assembly and budding of the M (membrane) and N (nucleoprotein) of SARS- CoV-2. In two specific aims, we will (i) determine the cellular and biophysical mechanisms by which SARS-CoV- 2 M form virus particles in silico, in vitro and in human cells and (ii) determine how N lipid binding drives localization that contributes to formation of new viral particles. These studies will be integrated with structural biology of M (Browhan laboratory) and N (Ollmann Saphire laboratory) and also be validated with authentic SARS-CoV-2 in a BSL-3 facility in collaboration with the Kuhn laboratory. These questions will be studied in a tightly integrated approach using structural and in vitro quantitative techniques to assess lipid-protein and protein-protein interactions and cellular assays to tease apart the molecular underpinnings of viral protein interactions necessary for viral budding and infection. Computationally, we will use coarse-grained (CG) molecular dynamics (MD) simulations to characterize the assembly process on the membrane and to identify a set of models for further refinement through all-atom (AA) MD simulations. This innovative and integrated approach will not only provide careful validation of the results, but also provide detailed structural insights into the lipid-protein and protein-protein interactions governing the assembly and budding of SARS-CoV-2. The protein interfaces of M and N identified in these studies, which will be key for virus assembly and spread, will inform future drug targeting against SARS-CoV-2 and other coronaviruses.