University Of Illinois At Chicago

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT This application represents a training plan designed to provide mentoring, career development, and support to the applicant as a clinician-scientist seeking to move research from the benchtop to the bedside in craniofacial and oral sciences. The training plan encompasses laboratory experimentation and professional and career development opportunities, and the plan is supported by the outstanding local and institutional resources available at UIC. The proposed research will address an important unmet clinical need facing craniofacial trauma. While the mandible is the strongest and largest facial bone, there is a high level of incidence for mandibular fractures. Unstable mandibular fractures exhibit delayed healing compared to fixed fractures, and their healing involves a chondrocyte-to-osteoblast developmental pathway that is not yet fully understood. Understanding the specific molecular pathways that control fracture resolution is important for improving clinical outcomes and the development of new therapeutics. The focus of this study is on a transmembrane proteoglycan, NG2/CSPG4. This molecule has been implicated in the mechanical response of mandibular chondrocytes in the temporomandibular joint and the progression of osteoarthritis, but it has not been studied in the context of endochondral fracture healing. The research plan in this proposal utilizes a preclinical murine model of endochondral fracture healing in the mandible, together with transgenic knockout animal models, to define the role of NG2/CSPG4 in the cell differentiation cascade that is required for the successful mineralization of a fracture callus. The proposed research plan will test the central hypothesis that mechanical loading-dependent NG2/CSPG4 signaling regulates the differentiation of osteochondral progenitor cells during endochondral ossification in mandibular fractures. Long-term, our goal is to understand how cells make decisions about their fate during bone regeneration. Aim 1 will evaluate the role of NG2/CSPG4 in the ability of osteochondral progenitor cells to differentiate into cartilage. Aim 2 will focus on the role of NG2/CSPG4 in the ability of cartilage cells to undergo mineralization. Together, the data generated from this project will address an important gap in knowledge surrounding mandibular fracture healing and bone biology more broadly and may identify a new therapeutic target for clinical intervention.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT To date there is no effective treatment for Alzheimer’s disease that decreases cognitive decline. Although the available drugs are effective at reducing amyloid beta, a multi-target drug approach is more likely to succeed in impacting cognitive function. One potentially complementary treatment approach is modulation of the autophagic lysosomal pathway (ALP), which is responsible for protein turnover and has been shown to regulate degradation of misfolded proteins in the CNS. Autophagy activation normally helps clear protein aggregates and damaged organelles via sequestration into autophagosomes, these vesicles eventually fuse with lysosomes, which then degrade the autophagic cargo. This pathway is more complicated in long-lived, polarized neurons, where autophagy largely initiates in the distal axon and thus autophagic lysosomal intermediates must be removed from the axon via retrograde transport to the soma where proteolytically active lysosomes reside. Disruptions in the transport or maturation of ALP intermediates has long been implicated in AD pathogenesis, as they buildup in dystrophic neurites around Aβ aggregates and likely contribute to Aβ production in AD. Preliminary data in our AD model of iPSC-derived human i3Neurons shows that ALP intermediates that accumulate in axonal swellings are cleared upon treatment with a novel compound identified in a high throughput screen as an autophagy upregulator in Hela cells. Importantly, our data shows that this compound also reduces both intracellular and extracellular Aβ42 and increases neuronal autophagy (increased LC3II/LC3I) in our AD model i3Neurons. Exciting new data from the Aldrich lab has identified lysosomal membrane protein 1 (LAMP1) as a direct target of the compound. Given these preliminary studies, we hypothesize that the novel compound, through direct interaction with LAMP1 and potential stabilization of LAMP1 interactions with retrograde machinery, increases retrograde lysosomal transport and autophagosome maturation, and thus clears axonal ALP vesicles and ultimately lowers Aβ levels. We will test this hypothesis by determining the mechanism by which the novel compound alters ALP transport and ALP composition and function, as well as identify new potential targets in neurons (by an unbiased proteomics approach). Through these studies, we will also determine if the FDA-approved drug Rapamycin can alter axonal ALP buildup or Aβ42 levels in comparison to the novel compound, thus shedding new insight into the translational potential of both drugs. Lastly, we will determine if the novel compound can reduce Aβ42 in a familial AD model and how it alters ALP in these neurons. Given the strong evidence that this compound can modulate axonal ALP transport, Aβ42 and neuronal autophagy, the results from the proposed experiments could be relevant to therapeutic approaches in other neurodegenerative diseases that have ALP dysfunction as a contributing pathological feature, such as Parkinson’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Dementia is a major global health challenge that lacks effective treatment and early diagnosis tools. Alzheimer’s disease (AD) comprises 70% of all dementia syndromes. The Lancet Commission recently urged a life-course model of AD prevention, providing impetus for the development of scalable early biomarkers. Mitochondria play a critical bioenergetic role in maintaining physiologic homeostasis, particularly for high energy demand organs, like the brain. Mitochondrial DNA (mtDNA) copy number (mtDNA-CN), a quantitative indicator of mitochondrial function, is strongly associated with AD in older adults. Growing evidence also implicates mtDNA mutation load, or mtDNA heteroplasmy (mtDNA-Het), in AD. Despite the accumulating evidence for a key role of these blood indicators in cognitive decline and AD in older adults, there is a paucity of research examining their relationships in midlife, a critical time when preventive interventions may be most effective. Moreover, the relationships between these mtDNA alterations and early emerging AD-related neurobiological substrates is unclear. While cardiovascular disease (CVD) risk factors have also been associated with mtDNA alterations, the temporal associations are not fully discerned. Whether mtDNA alterations could mediate the well-known but less well understood associations of heart and brain health is unknown. Our central hypothesis is that mtDNA alterations are associated with cognitive decline and AD-related neurobiological substrates in midlife and mediate the associations of early life CVD risk factors with midlife brain health. To test this hypothesis, we will leverage life- long measures of CVD risk factors and two midlife measures of cognitive function in the full Bogalusa Heart Study (BHS) cohort (N=1,298; 850 whites and 448 Blacks), along with AD-related neurobiological substrates from brain magnetic resonance imaging (MRI) and photon emission tomography (PET) scans available in a large subsample at midlife (N=700). Within the BHS, we further propose measurement of mtDNA alterations at two midlife time-points. Our well-powered validation effort will be conducted among diverse participants from the Trans-Omics for Precision Medicine program (N=3,724) with existing data. These resources will allow us to examine the prospective and temporal associations of mtDNA alterations with cognitive decline (Aim 1) and neurobiological substrates in midlife (Aim 2); and assess prospective and temporal associations of early life CVD risk factors with mtDNA and investigate mediating effects of mtDNA on associations of childhood CVD risk factors with midlife brain health (Aim 3). Our work could have broad impacts on population-wide and targeted efforts to curb dementia, informing drug development and risk stratification. The K99 training will allow me to conduct the first study examining prospective associations of midlife mtDNA with cognitive decline. Mentored by a team of experts in epidemiology, genomics, and neurobiological aging and led by my primary mentor Dr. Kelly, this award will undoubtedly accelerate my career independence in multi-omics research of brain aging.

NIH Research Projects · FY 2025 · 2023-08

The Center for Health in Cognitive Aging (CHECA), a new Alzheimer’s-related RCMAR at University of Illinois Chicago (UIC), will establish research and mentoring infrastructure for early-career faculty (RCMAR Scientists) from various backgrounds. The thematic focus of CHECA is behavioral and social pathways to Alzheimer’s Disease and Alzheimer’s Disease Related Dementias (AD/ADRD) in the context of dementia care and caregiving. Behavioral and social pathways to AD/ADRD involve multiple levels, from biobehavioral and psychological factors to social structures (e.g., health systems, social norms). The team-based research community will support Scientists to conduct pilot studies. CHECA will support research that (1) advances our understanding of environmental, sociocultural, and behavioral contributors of disparities in AD/ADRD; (2) develops or tests health-promoting interventions or resources (e.g., physical activity programs, technologies, social support from communities or healthcare providers); and (3) cross-fertilizes population science and health promotion approaches to address health disparities in adults aging with or at risk for AD/ADRD. CHECA will promote ADRD research at UIC by building behavioral and social research infrastructure (Aim 1), broadening the AD/ADRD research workforce through mentorship and training for early-career Scientists (Aim 2), and supporting pilot research (Aim 3). CHECA is committed to understanding and addressing health disparities from various perspectives, paying special attention to intersecting factors (e.g., living with a disability) and structural environmental, biological, and other contextual factors (e.g., healthcare systems) that interact in additive or nonadditive ways to contribute to ADRD etiology and health outcomes over the life course. The Leadership and Administrative Core (LAC) will provide scientific and intellectual leadership, strategic direction, and administrative oversight to produce synergy across CHECA Cores. The Research Education Component (REC) will provide a mentoring program for RCMAR Scientists to support their long-term careers, will implement the pilot funding program (solicitation, review, and selection of pilot studies for funding), and will provide tools and training relevant to the CHECA themes. The Analysis Core (AnC) will support Scientists’ pilot projects through mentoring and consultation, enrich methodological infrastructure, and promote collaboration among CHECA Scholars, mentors, and the research community. The Community Liaison Recruitment Core (CLRC) will provide CHECA Scientists and their mentors with training opportunities and resources for conducting community-engaged research and will facilitate bi-directional collaborations between CHECA and community partners. UIC provides a fertile ground for a new AD/ADRD RCMAR that joins population science and health promotion research to better understand and address ADRD-related health disparities.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY A significant number of individuals struggle to maintain sobriety as a result of withdrawal symptoms that emerge during early abstinence from alcohol. Despite this, pharmacotherapeutics for the treatment of withdrawal are lacking, at least in part, due to poor understanding of the mechanisms underlying symptoms of withdrawal. The rostromedial tegmental nucleus (RMTg) is a GABAergic region that exerts inhibitory control over midbrain bioaminergic and cholinergic nuclei. Recently, work from our lab revealed significantly enhanced cFos expression, a marker of recent neuronal activity, in the RMTg of chronic intermittent ethanol (CIE) exposed rats 12 hours into acute withdrawal. In addition, we showed that pharmacological inhibition of the RMTg attenuates withdrawal-induced anxiety-like behavior. However, the precise neural circuits that drive this putative RMTg hyperactivity is unknown. The lateral habenula (LHb) provides dense glutamatergic input to the RMTg and exhibits significant functional overlap with the RMTg. In addition, our preliminary data reveals a significant increase in cFos expression in RMTg-projecting lateral habenula (LHb) neurons during acute withdrawal. Together, these data lead us to hypothesize that LHb inputs to the RMTg play a mechanistic role in regulating symptoms of withdrawal from chronic ethanol exposure. Experiments in the current proposal are designed to test this hypothesis by using in vivo chemogenetics to bidirectionally and selectively manipulate LHb-RMTg activity during behavioral tests of withdrawal symptoms including anxiety-like behavior, mechanical pain sensitivity, and impulsive choice. Additional work will use ex vivo slice electrophysiology to explore the physiological neuroadaptations that occur in this circuitry during withdrawal from chronic ethanol exposure. Findings from these experiments will shed new light onto the role of this neural circuit in regulating symptoms of withdrawal and by extension have the potential to uncover new neurobiological targets for the treatment of alcohol use disorder.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Dementia is a major global health challenge that lacks effective treatment and early diagnosis tools. Alzheimer’s disease (AD) comprises 70% of all dementia syndromes. The Lancet Commission recently urged a life-course model of AD prevention, providing impetus for the development of scalable early biomarkers. Mitochondria play a critical bioenergetic role in maintaining physiologic homeostasis, particularly for high energy demand organs, like the brain. Mitochondrial DNA (mtDNA) copy number (mtDNA-CN), a quantitative indicator of mitochondrial function, is strongly associated with AD in older adults. Growing evidence also implicates mtDNA mutation load, or mtDNA heteroplasmy (mtDNA-Het), in AD. Despite the accumulating evidence for a key role of these blood indicators in cognitive decline and AD in older adults, there is a paucity of research examining their relationships in midlife, a critical time when preventive interventions may be most effective. Moreover, the relationships between these mtDNA alterations and early emerging AD-related neurobiological substrates is unclear. While cardiovascular disease (CVD) risk factors have also been associated with mtDNA alterations, the temporal associations are not fully discerned. Whether mtDNA alterations could mediate the well-known but less well understood associations of heart and brain health is unknown. Our central hypothesis is that mtDNA alterations are associated with cognitive decline and AD-related neurobiological substrates in midlife and mediate the associations of early life CVD risk factors with midlife brain health. To test this hypothesis, we will leverage life- long measures of CVD risk factors and two midlife measures of cognitive function in the full Bogalusa Heart Study (BHS) cohort (N=1,298; 850 whites and 448 Blacks), along with AD-related neurobiological substrates from brain magnetic resonance imaging (MRI) and photon emission tomography (PET) scans available in a large subsample at midlife (N=700). Within the BHS, we further propose measurement of mtDNA alterations at two midlife time-points. Our well-powered validation effort will be conducted among diverse participants from the Trans-Omics for Precision Medicine program (N=3,724) with existing data. These resources will allow us to examine the prospective and temporal associations of mtDNA alterations with cognitive decline (Aim 1) and neurobiological substrates in midlife (Aim 2); and assess prospective and temporal associations of early life CVD risk factors with mtDNA and investigate mediating effects of mtDNA on associations of childhood CVD risk factors with midlife brain health (Aim 3). Our work could have broad impacts on population-wide and targeted efforts to curb dementia, informing drug development and risk stratification. The K99 training will allow me to conduct the first study examining prospective associations of midlife mtDNA with cognitive decline. Mentored by a team of experts in epidemiology, genomics, and neurobiological aging and led by my primary mentor Dr. Kelly, this award will undoubtedly accelerate my career independence in multi-omics research of brain aging.

NIH Research Projects · FY 2026 · 2023-08

Non-alcoholic steatohepatitis (NASH) is emerging as a major worldwide cause of liver disease in children and adults. Recent studies demonstrate substantial heterogeneity in the phenotype of macrophages (MFs) infiltrating the liver during NASH. Recruited MFs exist as two subsets, with distinct activation states: (1) those closely resembling homeostatic Kupffer cells (KCs), or (2) lipid-associated MFs (LAMs). In 2020, a cluster of LAMs with high expression of osteopontin (OPN) was identified in livers with NASH. Notably, LAMs are differentially activated, compared to resident- and monocyte-derived KCs, and with a distinct ability to metabolize lipids. However, whether they also crosstalk with neighboring cells, to prevent steatosis and fibrosis in NASH, remains unknown. While recruitment of MFs into the liver, and subsequent activation, are considered proinflammatory and profibrotic events, currently a significant knowledge gap is a lack of understanding of whether OPNHigh MFs, are detrimental or protective in NASH. Preliminary data demonstrates that Spp1KI Mye are protected, whereas Spp1ΔMye have worse NASH activity scores than WT mice. Thus, our results suggest a paradigm shift, in that OPNHigh MFs may protect from NASH. Yet, the intercellular communication, and preventive and therapeutic potential of OPNHigh MFs, remain to be determined. Our overarching hypothesis is that OPNHigh MFs, by signaling to hepatocytes (HEPs) and hepatic stellate cells (HSCs), reduce liver steatosis and fibrosis, and protect from NASH. Aim 1 is to identify how OPNHigh MFs protect from steatosis. We hypothesize that OPNHigh MFs signal HEPs to upregulate arginase-2 (ARG2), which increases mitochondrial bioenergetics and fatty acid oxidation (FAO), reduces nitrosative stress, and protects from steatosis. To test this, first, we will identify the OPNHigh MF ‘secretome’ proteins that signal HEPs to upregulate ARG2; second, we will determine how the identified OPNHigh MF secretome proteins transactivate the ARG2 promoter, in HEPs; and third, we will elucidate how ARG2 increases mitochondrial bioenergetics and FAO, and reduces nitrosative stress, in HEPs. Aim 2 is to dissect how OPNHigh MFs protect from fibrosis. We hypothesize that OPNHigh MFs signal through secretome proteins to lower collagen-I, and/or modify the ‘matrisome’ landscape, to prevent fibrosis, and regulate cell behavior, in NASH. To prove this, first, we will identify the OPNHigh MF secretome proteins that signal HSCs to lower collagen-I deposition and/or increase its degradation; second, we will analyze whether OPNHigh MFs modify the matrisome landscape, and establish the OPNHigh MF-ECM correlation network; and third, we will perform liver scRNA-seq, and computationally identify how the OPNHigh MF-ECM network regulates cell behavior in NASH.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Labor migration is a major contributor to fueling the global AIDS epidemic and also the movement of HIV across country borders and populations. Migrants who inject drugs while in a host country are at especially high risk. Tajikistan, a small country in Central Asia, exports more than a million temporary labor migrants annually, many of whom inject drugs. These migrants are highly subject to the negative effects of intersectional stigma within Russia's cultural and socio-economic environment due to being both a migrant worker and a person who injects drugs (PWID). In addition, they are subject to extensive censure and marginalization from their non-drug-using Tajik peers in the close-knit diaspora communities in which they reside, especially if they acquire HIV. The adverse effects of stigmatization on those who experience it are well documented. The intersection of stigmatized identities as migrants and people who inject drugs (PWID), along with stigma associated with HIV infection, contributes to HIV risk behavior, poses as a barrier to accessing HIV testing and other prevention/treatment services, and results in poorer health outcomes for those living with HIV. The proposed study will investigate the character of stigma in the Moscow Tajik migrant community, and the effects of multiple intersecting forms of stigma on the health and well-being of Tajik labor migrants who inject drugs while living in Moscow (Aim 1). We will use the insight and findings gained through this formative research to develop an innovative intervention specifically designed to counter the negative effects of drug- related stigma within the Tajik migrant community that can affect HIV risk behavior and prevention among Tajik migrants in Moscow who inject drugs (Aim 2). The Stigma Reduction Intervention Approach Via Leaders of Diaspora (SRI-AVLOD) intervention will draw on the strengths of close-knit Tajik diaspora communities in which new norms and behaviors can be effectively diffused and promoted within and across migrant social networks if endorsed by leaders whom they trust. SRI-AVLOD is designed to recruit and train Tajik diaspora community leaders as agents of change and open up conversation within the Moscow Tajik community about the effects of drug and HIV-related stigma, to reduce stigmatizing beliefs and actions that negatively affect HIV risk behavior and prevention among community members who inject drugs. After developing a working prototype, we will deliver the intervention to small groups of Tajik migrant community leaders to assess its feasibility and acceptability, and need for further modifications (Aim 3). In future work we will deliver and test the efficacy of the SRI-AVLOD model in changing drug-related stigma and stigmatizing beliefs, behavior, and consequences at all levels (community, leadership, PWID) within the Tajik diaspora community and its positive effects in reducing HIV risk behavior and increasing the adoption of HIV prevention methods and services among its Tajik members who inject drugs.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The extracellular matrix (ECM) is a complex protein meshwork that constitutes the architectural scaffold of all tissues. In addition to its structural role, the ECM conveys biochemical signals to cells interpreted by receptors, like integrins, and controlling diverse cellular functions including adhesion and migration. The ECM is thus a key regulator of developmental processes and tissue homeostasis. Consequently, alterations in the composition and assembly of the ECM meshwork have been linked to a plethora of diseases including fibrosis, and cancer. Important progress toward understanding how the ECM meshwork is built and how the ECM govern cellular phenotypes have been made by studying major ECM proteins such as fibronectin and collagens. However, using sequence analysis, we have predicted that nearly 300 proteins can contribute to the ECM meshwork. Knowledge regarding the roles of these other ECM proteins remains preliminary. This represents a significant gap in our understanding of the ECM and in our ability to correct disease-causing ECM defects. We have recently become interested in one of these understudied ECM proteins, SNED1, after having found that it was associated with more highly aggressive breast cancers. Of clinical relevance, we found that higher SNED1 expression correlated with a worse prognosis for breast cancer patients. To gain insights into SNED1’s functions, we generated the first knockout (KO) mouse model of Sned1 and showed that Sned1 is an essential gene, since its KO resulted in early neonatal lethality due, in part, to craniofacial malformations. Importantly, we recently identified the first patients with SNED1 variants and they present with craniofacial malformations. Despite these observations, the mechanisms by which SNED1 contributes to embryonic development and cancer metastasis are unknown. Using novel tools we developed (antibodies, mouse models, cell lines, purified proteins), we have shown that SNED1 forms fibers within the ECM scaffold and contributes to its overall organization. In this proposal, we will test the hypothesis that SNED1 assembly in the ECM and its role in regulating ECM architecture depend on SNED1’s interactions with other ECM proteins and integrins. Leveraging our unique toolkit and combining our unique expertise in ECM protein biochemistry and ECM proteomics with state-of-the-art microscopy, we will conduct a time-resolved structure/function analysis to map which domains of SNED1 mediate its incorporation in the ECM (Aim 1); determine the role of ECM proteins/SNED1 interactions in SNED1 ECM assembly (Aim 2); and identify the SNED1 receptors at the cell surface governing SNED1-dependent ECM organization and responsible for the adhesive property of SNED1 (Aim 3)? Our goal is to fill critical gaps in our understanding of the fundamental mechanisms leading to ECM assembly, with respect to SNED1. This is a necessary step toward deciphering how perturbations of these mechanisms can lead to developmental defects and cancer progression. This work will also pave the way to the development of future therapeutic strategies to correct disease-causing alterations in ECM structure.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Retrieving biomedical articles from bibliographic databases requires accurate, detailed indexing of the topics that are discussed as well as their publication types and study designs. It is difficult for indexers to keep up with manual assignments in view of the explosion of published literature. Although NLM has recently employed automatic machine learning methods to index articles according to the major topics discussed, there is still no automatic means of indexing each article across all publication types and study designs. We have recently created a working prototype tool, Multi-Tagger, which has assigned probabilistic predictive scores for all PubMed articles for 50 different publication types and study designs (collectively, PTs). We now propose to develop Multi-Tagger 2.0, to handle a wider variety of study designs, articles, users and use cases, and to ensure that the data are disseminated in a form that is appropriate to each different type of user. Specifically, we aim to: Aim 1. Optimize methods for assigning Publication Types and study designs to both PubMed and non- PubMed biomedical articles, preprints and manuscripts. Aim 2. Evaluate PTs in detail, taking into account model performance, use cases and users. Aim 3. Optimize dissemination of PT predictive scores by query interface and API. Aim 4. Explore how to integrate Multi-Tagger with other tools for automating evidence synthesis. The proposed studies will greatly enhance retrieval of relevant articles and preprints across multiple databases, and will be useful for a wide range of biomedical end-users (clinicians, researchers, students and journal editors) as well as user groups including systematic review groups, bibliographic database managers, those studying preclinical animal models of human disease, and pharmaceutical companies developing new drug treatments. Improving the infrastructure of the biomedical literature will thus indirectly impact on human health.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), is a life- threatening disease that develops because of viral/bacterial infections (e.g. COVID-19), trauma, sepsis, pneumonia, or transfusion. ALI/ARDS remains a significant health burden in the United States with a high incidence and mortality rate (30-40%). Past interventional studies have mainly centered around early-stage supportive care such as ventilation which has limited utility. Despite several investigations, no effective pharmacological therapy exists for the treatment of ALI/ARDS. Mechanistically, ALI/ARDS is caused by the long- lasting activation of macrophages and infiltration of neutrophils due to their phenotypic shift towards the pro- inflammatory status. In the absence of ALI, alveolar macrophages (AMɸ) exist primarily in the anti-inflammatory phenotype, assisting in constructive processes. Upon sensing pathogens, AMɸ takes on an pro-inflammatory phenotype that secretes high levels of pro-inflammatory cytokines which can lead to an uncontrolled cytokine storm. We hypothesize that the acute pulmonary inflammation can be resolved by reprogramming macrophages by novel pharmacotherapies for effective treatment of ALI. Driven by this hypothesis, the goal of this project is to engineer an innovative macrophage-based pharmacotherapy (MEAT) for targeted immunomodulation in ALI. MEAT integrates macrophages’ inflammation targeting and therapeutic capabilities for ALI immune environment modulation. MEAT is composed of macrophages coated with a biomaterial that carries a pair of synergistic cytokines which can exert potent effects in programing macrophages and neutrophils to the anti-inflammatory phenotype. Two independent specific aims have been planned. In Aim 1, we will develop, optimize, and characterize MEAT capable of programming macrophages and neutrophils toward anti-inflammatory phenotypes in vitro. In this aim, we will determine the concentration-, ratio-, and temporal-requirements for optimal combination of IL-4 and IL-10 to program macrophages and neutrophils. We will then assemble and optimize MEAT to recapitulate these requirements for optimal macrophage and neutrophil modulation in vitro. In Aim 2, we will evaluate the efficacy of MEAT in resolving acute inflammation and thereby ALI. In this aim, we will evaluate i) MEAT’s capability to deliver synergistic cytokines to the lung tissues in the pre-clinical mouse model of ALI, and ii) the performance of MEAT in modulating the immune environment in inflamed lungs. Eventually, we will investigate the therapeutic efficacy of MEAT in alleviating ALI and restore normal lung functions. The PI (Early Stage Investigator) has assembled a team with complementary expertise to conduct this project. If successful, MEAT will revolutionize how ALI is treated and opens a new area for clinical research by unlocking unrealized applications of ALI cellular immunotherapies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary This K99/R00 Pathway to Independence Award's overall goal is to develop and assess the feasibility and preliminary efficacy of a web-based resilience-building intervention to address patients with advanced cancer and their family caregivers’ appraisals of advance care planning (ACP) and individual and dyadic coping. ACP is a process to facilitate decision-making for future medical care. By supporting individuals in understanding and sharing their values and goals, ACP can help patients meet their goals for health care treatment, decrease the emotional burden of decision-making for family caregivers, and increase satisfaction with end-of-life care and quality of death. Avoidance of ACP can delay the introduction of palliative and hospice care and risk patients receiving costly and aggressive care that not only is goal- discordant but also increases family caregivers’ anxiety and depression. The rationale for a web-based resilience-building intervention is that patients and family caregivers can be empowered to accurately appraise and use appropriate coping strategies for ACP by increasing their resilience skills. Building resilience has shown promising results in reducing cancer distress in adolescents and young adults with cancer and their parents. The proposed study is guided by the Systemic Transactional Model and utilizes a dyadic intervention approach. The training goals of the proposed study are to (1) develop expertise in dyadic study design and intervention development for people with cancer and their caregivers; (2) gain specialized knowledge in digital positive psychology interventions; (3) gain skills in leading randomized controlled trials and dissemination and implementation science; and (4) secure a tenure-track position. The Specific Aims for the K99 phase are to (1) identify the ACP deliberation process among 20 dyads of patients with advanced cancer and family caregivers and (2) conduct usability testing among 9 dyads to refine the content and design of the web-based resilience-building intervention. The Specific Aims for the R00 phase are to (1) assess the feasibility and acceptability of the web-based resilience-building intervention among patients with advanced cancer and their family caregivers through a randomized clinical trial in a cancer center setting; (2) evaluate the preliminary efficacy of the intervention compared to usual care on changes in the completion of advance directives (primary outcome) and on patient and caregiver resilience, individual and dyadic coping, anxiety, and depression (secondary outcomes); and (3) explore the dyadic effects of resilience within patients with advanced cancer and their family caregivers on individual and dyadic coping, anxiety, and depression, using the Actor-Partner Interdependence Model. The expected outcomes are identification and development of the essential components of the web-based intervention (K99 phase) and data that provides a strong foundation for further development of the intervention for R01 applications and future research (R00 phase).

NIH Research Projects · FY 2025 · 2023-08

This project aims to assess the feasibility and preliminary efficacy of a web-based resilience-building intervention to address advance care planning (ACP) and dyadic coping among people with advanced cancer and their family caregivers. ACP is a process to facilitate decision-making for future medical care. Helping patients understand and communicate their values and goals with family caregivers can lead to care that aligns with patient wishes, reduce caregiver emotional burden, and improve patient satisfaction with end-of-life care and the quality of death. Avoidance of ACP can delay introduction of palliative and hospice care and lead to costly and aggressive goal-discordant care that also increases family caregivers’ anxiety and depression. Building resilience has shown promising results in reducing cancer distress and anxiety in adults with cancer and family caregivers. The rationale for the proposed web-based resilience-building intervention is that increasing resilience skills for ACP can empower patients and family caregivers to become aware of their resilience resources and use appropriate coping strategies to engage in ACP discussions and complete advance directives. Our preliminary qualitative data highlighted the characteristics of resilience needed during ACP discussions among people with cancer who have completed advance directives. Guided by this interview data and existing literature in positive psychology and dyadic intervention design, we developed a prototype of a web-based resilience-building intervention for patients with advanced cancer and their family caregivers. Participants with prior ACP experience endorsed the website for its simplicity, clarity, and ease of navigation without technical support and said that the intervention was easy to understand and helpful for facilitating ACP discussions and completing advance directives. Our next step is to assess the feasibility, acceptability, and usability of the intervention among 76 dyads of patients with advanced cancer and their family caregivers (Aim 1). We will conduct a randomized controlled trial in a cancer center setting to evaluate the preliminary efficacy of the intervention (n = 38 dyads) compared to usual care (n = 38 dyads) on changes in completion of advance directives (primary outcome) and on patient and caregiver resilience, optimism, dyadic communication and coping, knowledge, self-efficacy, anxiety, and depression (secondary outcomes) (Aim 2). We will conduct assessments at baseline and 8 weeks post-randomization. Finally, we will use the actor-partner interdependence model to explore the dyadic effects of resilience on self-efficacy, optimism, dyadic communication and coping, anxiety, and depression (Aim 3). The expected outcomes are demonstration of the web-based resilience-building intervention’s feasibility, acceptability, usability, and preliminary efficacy and data that provide a strong foundation for further implementation of the intervention for R01 applications and future research.

NIH Research Projects · FY 2025 · 2023-08

Viruses are inanimate biomolecular assemblages constructed inside host cells for the tasks of transmission and infection. Despite the high degree of fidelity required to produce functional infectious particles, viruses are adept at developing resistance to natural and vaccine-induced immune responses through escape mutations. This is due to the presence of multiple redundant self-assembly signals, resulting in a fitness landscape with vast regions of favorable sequence space. Viruses can sample extensively from this space to arrive at escape variants, while retaining the capacity to properly assemble. We propose a transformative approach that aims to target large regions of viral fitness landscapes, with the goal of overcoming antiviral drug resistance. In this approach, various viral self-assembly signals will be exploited to induce the mis-assembly of viral components toward non- infectious endpoints. Computationally designed peptides will steer the self-assembly process toward trapped states accessible to large numbers of genetic variants. Peptides are ideal for this task for a number of reasons. Firstly, peptides can target large surface areas of proteins, allowing for binding to targets that lack deep binding pockets. Secondly, peptides can be designed to self-assemble into diverse supramolecular structures, such as fibers, two-dimensional arrays, and liquid condensates. We will leverage the ability of peptides to form these types of structures to induce the formation of non- infectious viral mis-assemblies. Finally, peptides can be optimized for membrane permeability, allowing for the targeting of viral replication inside host cells. Drug-induced mis-assembly of HIV viral capsid has been suggested as one of several possible mechanisms of action of the small-molecule drug PF74. We aim to develop a rationally-guided design approach to enable the wide-spread application of this novel mechanism of anti-viral action. We will target three domains of the HIV Gag protein known to play distinct roles in viral assembly for directed mis-assembly by computationally designed peptides. The resulting peptides will incorporate design elements that direct the trapping of viral components within one-dimensional fibers, two-dimensional arrays, and three-dimensional liquid condensates. Self- assembly will be monitored at the single-molecule level using Interferometric Mass Spectrometry, and at the bulk level with Bio-layer Interferometry and Dynamic Light Scattering, while Electron Microscopy and Fluorescence Microscopy will be used to visualize the induced assemblies. This project paves the way for development of antiviral therapies through mechanisms that pose high barriers to antiviral resistance.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Hematopoiesis is a continuous process of blood-cell production occurring through the orchestrated activity of hematopoietic stem cells (HSCs). Although HSCs have tremendous clinical utility due to their ability to reconstitute the hematopoietic system by transplantation, their benefit remains limited by the lack of matched donors. Direct reprogramming of endothelial cells into HSCs via induction of reprogramming factors has recently emerged as a promising alternative. The overall goal of our proposal is to reveal the molecular mechanisms by which the reprogramming factors FOSB, GFI1, RUNX1, and SPI1 (FGRS) revert endothelial cells to functional reprogrammed HSCs (reHSCs). Understanding the basis by which the genetic networks become rewired for this profound cell type conversion will provide insights into diverse forms of reprogramming, development, and disease. We discovered that early in the reprogramming process, FGRS directly coordinate two tasks: selection and activation of multipotent HSC enhancers and disruption of endothelial enhancers and transcription factors (TFs). We hypothesize that the effect of FGRS on endothelial TF binding is as crucial for reprogramming as the activation of multipotency enhancers, and we propose to dissect the underlying molecular mechanisms for these processes. Using single-cell multiomic (scRNA & ATAC-seq) profiling, we further discovered that in intermediate reprogramming, the relatively homogenous starting endothelial cells are replaced by heterogeneous HSC populations. How the transition from somatic (endothelial) to multipotent (HSC) regulatory programs occurs in individual cells undergoing in vitro reprogramming remains unknown. To potentiate in vivo reprogramming, we generated a novel transgenic mouse model that allows constant FGRS expression in all somatic tissues and facilitates the recording of all key bifurcating events that lead to HSC establishment and maintenance. Based on our studies, we propose to dissect the molecular and cellular mechanisms by which FGRS promote cell fate changes in the context of endothelial-to-HSC reprogramming. In our first aim, we will uncover the molecular mechanisms by which FGRS target and modulate endothelial and HSC gene regulatory networks. In the second aim, we will delineate the intrinsic and extrinsic signaling pathways that promote endothelial-to-HSC reprogramming. We expect that our program will yield fundamental insights into the control of mammalian cell identity and may lead to novel strategies to generate therapeutically relevant HSCs with high efficiency.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Nanoparticle therapeutics (NTs) that encapsulate drugs in a nanoscale particle has emerged as a primising therapeutic modality for treating many diseases. NTs encompass a diverse array of nanoparticle types and can easily incorporate a wide-array of drugs, ranging from small molecules, to macromolecules, to biologics. The diversity of nanoparticles and encapsulated drugs renders NTs a versatile therapeutic modality that is being clincally investigated to treat many dieseases in various tissues. Like any other therapetic modality, the successful application of NTs requires their specific delivery to target sites while avoiding off-target accumulation. However, owing to their distinct features (e.g. large-size), NTs face unique biological barriers which lead to their unfavorable pharmacokinetics (PK), biodistribution, and pharmacodynamics (PD) profiles. As such, a pressing and unaddressed challenge is to better understand the biological barriers for NTs and to develop effective strategies to guide the precise delivery of NTs to unleash their full therapeutic potential. Toward this end, the overarching goal of my research program is to identify ideal delivery parameters for NTs and to develop novel strategeis for precise delivery of NTs. One strategy we are focusing on is to utilize inspirations from the intrinsic biology, living cells in particular. Indeed, living cells such as circulatory cells can be leveraged as ideal delivery systems. Circulatory cells can navigate the body, sense pathological signals, and reach diseased tissues via an active transport mechanism. NTs can be loaded inside or onto the surface of circulatory cells to be delivered to target sites. My research has made significant strides in this area where we have developed novel methods to incorporate NTs with diverse living cells and demonstrated that two circulatory cells (erythrocytes and macrophages) could modulate the PK, biodistribution, and efficacy of NTs. The rapid progression in advancing cells towards NTs delivery highlights the urgent need for mechanistic studies to i) elucidate how the interface between living cell carriers and NTs impacts the transport of NTs and migration of carrier cells and ii) to identify principles for utilizing living cells for precise delivery of NTs. We aim to capitalize our expertise in nanoparticle design and cell engineering to address this unmet need. Specifically, over the next five years, using inflammation that occurs in various tissues as a model, we will focus on i) understanding how cell-based carriers impact the outcomes of NTs delivery, ii) studying how the loading and physicochemical properties of NTs influence the carrier cells’ migration, and iii) developing multiscale strategies to achieve cell-specific delivery of NTs. These studies will enable us to establish a set of design rules that govern the delivery efficacy and interactions of NTs with living cells, which will ultimately improve the capability and broaden the spectrum of NTs for treating various diseases. Successful realization of our program will not only contribute to understanding the key features for a NTs to interact with the living cells but also develop a set of principles for rational engineering living cells to improve the biological outcomes of NTs and other therapeutics.

NIH Research Projects · FY 2024 · 2023-07

Abstract Approximately 20% of breast cancers detected through mammography are pre-invasive Ductal Carcinoma in situ (DCIS). If left untreated, approximately 20-50% of DCIS will progress to more deadly Invasive Ductal Carcinoma (IDC). No prognostic biomarkers can reliably predict the risk of progression from DCIS to IDC. Similar genomic profiles of matched pre-invasive DCIS and IDC suggests that the progression is not driven by genetic aberrations in DCIS cells, but microenvironmental factors, such as hypoxia and metabolic stress prevalent in DCIS, may drive the transition. We need innovative models to investigate how to halt steps of DCIS progression to invasive phenotypes and subsequent metastasis from the primary site. This proposal directly addresses this unmet need by developing a novel three-dimensional in vitro organoid model that recapitulates key hallmarks of DCIS to IDC progression: tumor-size induced hypoxia and metabolic stress, tumor heterogeneity and spontaneous emergence of migratory phenotype in the same parent cells without any additional stimulus. A tangible advantage of the proposed organoid models is the ability to precisely and reproducibly study how the hypoxic microenvironment induces tumor migration in real time and in isolation from non-tumor cells present in vivo, providing unique opportunity to define tumor-intrinsic mechanisms of DCIS to IDC progression. During July 2018-Feb 2022 ESI MERIT Award period, we have shown that inhibition of tumor-secreted factors effectively halts organoid migration, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. We have also analyzed secretome from metastatic breast cancer pleural effusion showing significantly higher levels of CCL2/MCP1, CXCL10/IP10, IL-6, IL-8, regulatory IL-10, and IL-7 and IL-15. Strategies to neutralize these key cytokines may generate anti-tumor responses in the pleural environment. Microarray analysis of hypoxia-induced migration and secretome-induced migration suggested role of Rho GTPase and PI3K/AKT signaling pathways in maintaining migration. Our results show that hypoxic organoid models exhibit partial EMT signatures as early as day 1, which is maintained as these non-migratory organoids transition to migratory phenotypes. During the two-year extension period, we will continue 1) to optimize our DCIS models incorporating ductal structure and other components from DCIS microenvironments; 2) to test new mechanisms linking tumor-intrinsic hypoxia, partial/hybrid EMT and collective migration; 3) to inhibit signaling mechanisms to halt emergence of migratory phenotypes. The successful completion of the proposed work will provide answers to two fundamental questions in the progression of invasive breast cancer: 1) What causes some DCIS cells to become migratory and develop into invasive tumors? 2) How and where does the migratory phenotype (IDC) emerge? The mechanistic understanding gained from these studies will improve diagnosis, lead to the development of treatment strategies to arrest invasion at the pre-malignant stage, and thus prevent patient overtreatment.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract The inflammatory conditions and the complex physiological changes associated with chronic immunological responses to wound healing in type 2 diabetes (T2DM) challenge the outcomes of the treatments for bone regeneration. The related morbidity and costs associated with bone healing failures are considerable and increasing. Recent discoveries have demonstrated that the immune system is tightly linked to bone physiology and that MSC derived extracellular vesicles (EVs) play a prominent role in immunomodulation of bone repair. While recombinant human bone morphogenetic protein 2 (rhBMP2) mediated bone regeneration is well established, the challenges of controlling inflammation arising from rhBMP2 usage is exacerbated in the presence of immunomodulating systemic diseases such as T2DM present a significant knowledge gap that limit clinical outcomes. Based on preliminary studies of inflammation informed MSC EV microRNA (miRNA) composition and effects on immunomodulation and bone regeneration, we hypothesize that a dual delivery of engineered MSC EVs with anti-inflammatory properties and rhBMP2 using a combinatorial delivery system with self-assembling leucine zipper (LZ) peptide-based hydrogels and alginate beads can enhance bone healing in T2DM. We propose three specific aims to test this hypothesis. In aim 1, we will test our preliminary data driven hypothesis that a subset of altered miRNAs in preconditioned MSC EVs contributes to the enhanced immunomodulatory function of the inflammatory preconditioned MSC derived EVs. We will also generate engineered MSC EVs that overexpress candidate miRNA with anti-inflammatory properties to further study the mechanistic aspects by which individual EV related miRNAs control the immunomodulation functionality. In aim 2, we will create a novel dual delivery system with LZ-hydrogels and alginate beads that contain engineered EVs and rhBMP2 respectively. We will test our hypothesis that 1) EVs with anti-inflammatory properties (generated from aim 1) can be released during the inflammatory phase of healing (up to 7 days) from a self-assembling LZ- hydrogel and 2) rhBMP2 that is encapsulated in alginate beads with an extended-release profile (4-6 weeks) can provide a long term osteoinductive signal to promote/enhance bone regeneration. In aim 3, using a diabetic mouse calvarial defect model, we will test the hypothesis that engineered MSC EVs containing anti-inflammatory miRNA will suppress inflammation upon wounding while sustained released rhBMP2 promote bone healing. Overall, the results of these first studies of the combinatorial usage of engineered and MSC EVs rhBMP2 will provide a foundation for continued definition of MSC EV-mediated immunomodulation, and the regenerative properties and applicability of the dual delivery system to bone healing in T2DM. Further, these studies will also provide a framework for the use of growth factors and engineered EVs in combination for tissue engineering applications.

NIH Research Projects · FY 2025 · 2023-06

Infectious agents have infected prokaryotes and eukaryotes throughout evolution. Indeed, there is co-evolution of organisms and their infectious agents, with development of protective responses in the hosts and adaptive countermeasures by the infectious agents. Infectious endemic retroviruses like murine leukemia virus (MLV) have existed in mice for millions of years and provide us with an outstanding model system to understand how mammalian hosts suppress virus replication and conversely, how viruses counteract this restriction. One system of viral restriction is conferred by the apolipoprotein B mRNA editing enzyme, catalytic peptide 3 (A3) family of proteins, which are packaged into retroviruses in virion-producing cells and after infection of target cells, either block reverse transcription or deaminate deoxycytidine residues in single-stranded DNA, resulting in uracils and G-to-A mutations in the viral genome. The products of retrovirus reverse transcription (ssRNA, ssDNA and dsDNA) are also sensed by host nucleic acid sensors. Sensor binding to viral nucleic acid leads to the production of anti-viral cytokines and chemokines, such as type I interferons, which “warns” surrounding cells to arm themselves against infection by producing proteins such as A3. These host anti-viral events are believed to occur largely in the cytoplasm, where A3 proteins and many host sensors are believed to function. Retroviruses enter cells when the viral and host membranes fuse and capsids are deposited in the cytoplasm. Reverse transcription initiates from within the capsid and capsid dissociation and reverse transcription are mutually dependent; because DNA is more rigid than RNA, without capsid dissociation, reverse transcription cannot proceed and conversely, the generation of DNA facilitates capsid dissociation. The reverse transcription complex not only consists of viral RNA, DNA and the viral proteins reverse transcriptase and integrase, but viral capsid and other proteins such as the MLV protein p12, which is needed for tethering of the proviral DNA to host chromatin to achieve integration. Recently, there has been much debate as to whether reverse transcription occurs solely in the cytoplasm or in the nucleus or both. Our lab pioneered the use of in vivo mouse models to study how A3 proteins restrict retrovirus infection and has used A3 and nucleic acid sensor knockout (KO) mice and genetically engineered animals that express human A3 proteins in these studies. Our data, based on our analysis of A3 KO mice and cells, suggest that the initial step occurs in the cytoplasm but that reverse transcription may also occur in the cytoplasm. With these mouse models, we have the tools to carry out in vitro, ex vivo and in vivo studies to determine how A3-mediated restriction and sensing of reverse transcripts are integrated with reverse transcription and nuclear entry for MLV and its natural host, the mouse. To accomplish this, we propose 3 aims, that will determine: I. Where in the cells APOBEC3 proteins block reverse transcription or deaminate viral DNA; II. What stage in reverse transcription and where in the cell the host base excision repair enzyme, UNG, removes uracil from APOBEC3G-deaminated viral DNA and the consequences of this for viral escape; III. Whether host sensing of viral nucleic acid takes place in the cytoplasm, nucleus or both compartments. Determination of when and where these events occur is critical to understanding how retroviruses, including HIV, evade host immunity and the identification of which steps are likely to be the best targets for interventional therapies at the early stages of infection.

NIH Research Projects · FY 2025 · 2023-06

Abstract Chemical warfare agents such as mustards and arsenicals cause skin damage characterized by erythema, inflammation, and skin blistering, followed by a prolonged healing period. Although inflammatory cells and related cytokines are known to be increased in skin exposed to both mustards and arsenicals, similarities and differences in the roles of subsets of these cells in mustard- or arsenical-induced skin damage, as well as subsequent healing, remain to be elucidated. A goal of this proposal is to develop capacity to study the role of the inflammatory response, particularly the macrophage response, using cutting edge single cell techniques, in mustard and arsenical skin injury. This will include generating preliminary data with prototype agents and planning for experiments with restricted agents to support future NIH CounterACT grant submissions in the following aims: for Specific Aim 1, we will define the response of wound inflammatory cell subsets to vesicant skin injury using nitrogen mustard (NM) as prototype mustard and phenylarsine oxide (PAO) as prototype arsenical. For Specific Aim 2, since we and others have identified NLRP-3 is a key regulator of the inflammatory response to skin injury, we will determine the role of the NOD-Like Receptor (NLRP)-3 inflammasome in vesicant skin injury and repair. For Specific Aim 3, we will develop a relationship with MRIGlobal to perform studies with restricted vesicants. MRIGlobal is a contract research organization whose laboratory is approved for use of more potent restricted vesicants. Successful completion of this project will 1) bring a new skin wound healing research group into the chemical countermeasure field, 2) improve understanding of the role of inflammatory cell subsets in the response to vesicant injury, 3) establish the importance of the NLRP-3 inflammasome in regulating this inflammatory response and test an NLRP-3 inhibitor as a potential countermeasure and 4) develop a relationship with MRIGlobal for future studies using restricted vesicants. Ultimately, we plan to use knowledge generated by our research to develop novel countermeasures that target the inflammatory response.

NIH Research Projects · FY 2026 · 2023-06

Project Summary and Relevance. Hepatitis B virus (HBV) infection is a worldwide health problem. It is estimated that there are 200 to 500 million HBV chronic carriers in the world for whom, to date, there is no reliable treatment. HBV causes both acute and chronic liver disease and is responsible for an estimated one million deaths annually. Currently available therapies reduce viral loads but fail to resolve chronic HBV infections. Therefore, effective treatments for chronic HBV infection are urgently required. The major obstacle to the resolution of chronic HBV infections is the eradication or inactivation of nuclear HBV covalently closed circular (ccc) DNA which is the template for viral transcription. To this end, we have developed HNF1α-null HBV transgenic mice and liver-specific Tet-deficient HBV transgenic mice. HNF1α-null HBV transgenic mice synthesize nuclear HBV cccDNA, a fraction of which is extensively methylated in adult mice. Liver-specific Tet-deficient HBV transgenic mice are viable, essentially display normal liver physiology, but lack detectable HBV transcription and replication (i.e. they are effectively “cured”) suggesting Tet is also essential for viral biosynthesis. The observation that Tet-deficient HBV transgenic mice fail to support HBV biosynthesis is consistent with the suggestion that Tet is essential for the developmental demethylation of HBV genomic DNA which epigenetically governs HBV transcription by modulating viral chromatin structure in vivo. Defining the precise temporal requirements for Tet expression associated with HBV transcription and replication will indicate the liver developmental stages when viral biosynthesis is susceptible to inhibition by Tet deficiency. This will be achieved by modulating Tet expression using our recently developed tamoxifen-inducible Tet-deficient HBV transgenic mouse model system. Using this system, the developmental control of HBV transcription, viral biosynthesis and HBV DNA methylation by Tet expression will be established and correlated with the epigenetic histone marks and chromatin structure associated with the HBV genome. Similar studies will be performed using the HNF1α-null Tet-deficient HBV transgenic mouse model of chronic viral infection so the developmental control of HBV transcription, viral biosynthesis, HBV DNA methylation, epigenetic histone marks and chromatin structure by Tet expression associated with the HBV genome can be compared between the HBV transgene DNA and the nuclear HBV cccDNA. Finally, the development of HBV transgenic mice supporting viral biosynthesis exclusively from extrachromosomal genomic DNA, a more physiologically relevant mouse model of chronic viral infection, will be developed so the effect of Tet-deficiency on HBV biosynthesis derived solely from HBV cccDNA can be determined. Defining the molecular signals responsible for the loss of HBV biosynthesis due to Tet deficiency may lead to the identification of cellular therapeutic targets, including but not restricted to Tet proteins, that are amenable to the development of novel small molecular weight therapeutic inhibitors to resolve rather than simply treat chronic HBV infection.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract This four-year proposal for the K08 Mentored Clinical Scientist Career Development Award aims to further the professional skills of the candidate Daniel E. Maidana, MD, PhD while addressing critical scientific inquiries related to the contribution of mononuclear phagocytes to photoreceptor (PR) cell death in retinal detachment (RD). The candidate's proposed career and scientific goals rely on the protected research time necessary to master advanced laboratory methods and develop leadership skills under the guidance of an expert multidisciplinary mentoring team. This collaborative work, which builds on prior research and established mentoring relationships, will provide the basis for a successful and productive transition to independence. The career advancement plan for the candidate, currently an Assistant Professor of Ophthalmology at the University of Illinois at Chicago (UIC), will consist of i) graduate-level courses in immunology, biostatistics, transcriptomics, and bioinformatics, at UIC; ii) advanced laboratory technical and analytical methods, guided by an expert Mentoring and Advisory Committee; iii) development of management and mentoring skills required to lead a productive, independent laboratory. The institutional environment, departmental support, and cross- disciplinary mentorship team will enable the candidate to maximize productivity during these four years. The Department of Ophthalmology and Visual Sciences at UIC has a consistent record in transitioning early physician-scientists to established independent investigators and strongly supports the candidate for this award. The scientific goal of this proposal is to define the independent contribution of retinal microglia (MG) and blood- derived monocytes/macrophages (Mø) to PR demise and vision loss in an experimental model of RD. Since MG and Mø can either contribute to or resolve the initial injury, several therapeutic approaches have been recently proposed to modulate these cells. However, recent work has unveiled technical limitations and a lack of specificity in the methods used to ablate MG and Mø, thus limiting our understanding of their independent role in promoting or reducing PR cell death. This goal will be accomplished using a novel inducible conditional deletion model to i) define the role of MG in dead PR clearance in early RD; ii) dissect the contribution of MG and Mø phenotypes to promote PR demise in late RD; and iii) determine the neuroprotective potential of MG and Mø to rescue PR cell death following RD. The successful completion of this proposal will generate technical and scientific advancements in our understanding of MG and Mø. We expect this work to provide mechanistic insights to develop effective neuroprotective therapies, allowing us to maximize visual outcomes in the detached retina to prevent vision loss.

NIH Research Projects · FY 2024 · 2023-05

Project Summary In 2020 alone, about 200 million oral antibiotic prescriptions were dispensed by outpatient healthcare providers in the United States. Broad-spectrum antibiotics make up most of these prescriptions. However, in addition to targeting the intended pathogen, broad-spectrum antibiotics alter the composition of the human microbiome (dysbiosis). Dysbiosis caused by broad-spectrum antibiotic treatment can increase susceptibility to or worsen the outcome of disease, including inflammatory bowel disease, cancer, and psychiatric and neurodegenerative disorders. Therefore, it is important to develop therapeutic strategies that minimize the collateral damage of broad-spectrum antibiotics. Here we propose to explore molecules that selectively antagonize the activity of broad-spectrum antibiotics as a strategy to minimize dysbiosis. The advantage of selective antidotes over the alternative strategy of developing narrow-spectrum or pathogen-targeted antibiotics is to be able to continue to use already developed broad-spectrum antibiotics. Proof-of-concept for the selective antidote strategy has been recently provided. From the four previously identified antidotes, three are not available in the US due to their toxicity, and the remaining one has poor water solubility and unfavorable pharmacokinetics. We have recently discovered a family of depsipeptide natural products we named pseudovibriamides. Pseudovibriamides are produced by Pseudovibrio bacteria that are part of the healthy microbiome of marine sponges. Interestingly, Pseudovibrio has been proposed to contribute to marine sponge health by producing broad-spectrum antibiotics that can prevent the growth of pathogens. Marine sponges are ancient animals that form important symbiotic relationships with their microbes. Thus, broad-spectrum antibiotics known to be produced by Pseudovibrio and other sponge bacteria would cause dysbiosis in the sponge animal. We hypothesize that pseudovibriamides act as selective antidotes to protect commensal bacteria and the sponge host but allow activity against pathogens. We envisage the ecological role of pseudovibriamides may be translated into pharmaceutical applications. This proposal has two aims. Aim 1 is to improve access to pseudovibriamides using biosynthetic methods and Aim 2 is to explore their antibiotic and strain spectra. We will use sponge bacteria commensals and pathogens as controls to test the hypothesis. We will then explore the spectrum of antidote activity of pseudovibriamides with widely used antibiotics and the human pathogens they are intended to treat, and with prevalent and abundant human commensals. Thus, this proposal will enable facile access to and will explore the antidote spectrum of a family of bacterial natural products shown to have antibiotic antidote activity. What we learn will serve as steppingstones for future studies on their mode of action to enable the discovery of further antidotes.

NIH Research Projects · FY 2025 · 2023-05

Metastasis is the most common cause of breast cancer mortality. Of the breast cancer subtypes, triple- negative breast cancer (TNBC) is the deadliest due to its increased likelihood to metastasize. Tumor cell extravasation is a critical step of metastasis and allows circulating tumor cells to exit the vasculature and seed distant tissues. Clear understanding of the major regulators of tumor cell extravasation will provide insights into the progression of TNBC metastasis. One potential regulator of TNBC cell extravasation is ACKR1. In many contexts, ACKR1 expression is required in endothelial cells (EC) for leukocyte extravasation. Endothelial ACKR1 binds CXCL2, a promigratory chemokine, and localizes it to EC junctions to guide neutrophils through leukocyte extravasation. CXCL2 expression in TNBC cells is also necessary for tumor cell extravasation from lung microvasculature and for tumor metastasis. Our preliminary data show that ACKR1 is required in at least one stromal cell type for TNBC metastasis from the primary tumor to the lung. These data suggest that endothelial ACKR1-CXCL2 interactions may mediate tumor cell extravasation in TNBC metastasis. Therefore, we hypothesize that endothelial ACKR1 promotes TNBC cell extravasation by retaining CXCL2 at EC junctions, resulting in the increased metastatic spread of TNBC. To address this hypothesis, we will examine the in vivo significance of endothelial ACKR1 expression using our validated ACKR1 endothelial cell-specific knockout mouse model. We will test the requirement for endothelial ACKR1 for metastasis of orthotopically implanted TNBC tumors to distant sites in the lung and for extravasation of circulating tumor cells into lung tissue. We will determine which steps of extravasation require ACKR1 by evaluating ACKR1-low and ACKR1-overexpressing ECs using an Ibidi flow co-culture system that recapitulates the shear stress conditions of pulmonary microvasculature. We will examine whether these steps are dependent on CXCL2 by introducing CXCL2-neutralizing antibodies to the Ibidi flow system and observing their effects on each extravasation step. Our proposed studies will establish the role of endothelial ACKR1 in TNBC metastatic progression and determine the specific steps of tumor cell extravasation in which endothelial ACKR1 and CXCL2 function. Understanding these processes may guide development of ACKR1 as a prognostic marker for metastasis and can provide mechanistic insight into candidate chemokine and chemokine receptor inhibitors under evaluation for treatment of breast cancer.

NIH Research Projects · FY 2026 · 2023-05

7. Abstract Nearly half (47%) of people with end-stage kidney disease (ESKD) whose kidney function is restored after kidney transplantation experience chronic pain compared to 19% of adults in the US general population. Pain is associated with comorbid fatigue, depression and anxiety, and withdrawal from usual physical and social activities; resulting in an inability to participate in and enjoy life. Severe pain can result in nonadherence to immunosuppression and treatment protocols and result in an increased risk of rejection, graft loss, and mortality. The role of symbiotic microbes (microbiota) in the gastrointestinal tract, and their functional genes (microbiome), is well established in diseases involving pain. Diet and stress play a major role in synthesis of signaling molecules critical to immunologic, metabolic, and endocrine pathways regulating chronic pain. Dietary patterns change dramatically after transplantation, as recipients move from a restricted “renal” diet to a regular diet, often resulting in increased consumption of foods high in sugars and fat. Moreover, psychological stress significantly impairs the function of the microbiome, initiating biological pathways involved in pain, leading to a disproportionate pain burden. Because the microbiome, serum metabolites, and pain are dynamic, our novel investigation will employ a prospective repeated measures design to interrogate the dynamic temporal relationships between the microbiome, metabolites associated with pathways regulating pain, transplantation factors (e.g. immunosuppression, kidney function), changing dietary patterns, and perceived stress, on pain scores before and after kidney transplantation. We posit the gut microbiome, and its byproducts, may partially explain the underlying biological mechanisms of pain Interference in kidney disease. We will address three aims: 1) To determine differential dynamic temporal relationships between microbial composition/functional genes and circulating serum metabolites in KTRs with pain vs no pain, 2) To determine the moderation effects of diet and perceived stress on dynamic temporal relationships between microbiome features, serum metabolites, and pain scores among KTRs, and 3) To use machine learning algorithms to identify host-microbial interactions that are causally linked to pain interference among KTRs. Because kidney function is restored, the kidney transplant model is powerful to study the longitudinal relationships between the microbiome, circulating metabolites and chronic pain in people with ESKD to develop patient-centered interventions to treat pain across the spectrum of CKD.