University Of Alabama At Birmingham

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Decades of global effort have yet to result in a robust and effective preventative vaccine for HIV, although it has yielded remarkable insights into the likely requirements for immunogen design and potential correlates of vaccine mediate protection. The encouraging results of RV144 unfortunately were not reproduced in its adaptation to South Africa with HVTN 702, however both have demonstrated that a finely tuned antibody (Ab) response to gp120, particularly V1V2 will likely be key to an effective vaccine. Similarly, the VRC01 efficacy trial established the defining threshold for a gp120 CD4 binding site broadly neutralizing Ab to mediate protection, and numerous highly engineered gp120-based immunogens in clinical trials attempting to induce precise germline and B cell receptor development pathways, demonstrate the necessity of clearly defining gp120 specific B cell memory development. Development of a protective Ab response to HIV is likely dependent on the characteristics of the pre-existing B cell population present prior to HIV infection or vaccination, referred to as pre-immune B cells. We and others have demonstrated increased HIV-specific plasma Abs in individuals at high-risk for HIV acquisition (HR) and recently we have demonstrated that HIV gp120-specific pre-immune B cells include as expected naïve B cells, but also a substantial non-naïve proportion consisting of IgA, IgG, and IgM memory B cells. Following HIV Env vaccination, both pre-immune B cell populations respond and undergo somatic hypermutation and affinity maturation to give rise to high affinity HIV Env-specific Abs. Although the composition of the naïve pre- immune HIV Env-specific repertoire is driven by germline genetics and likely to be highly similar amongst individuals, the composition of non-naïve pre-immune HIV Env-specific repertoire is likely to be more heterogenous. Its composition is likely driven by exposure to cross-reactive antigens such as microflora and potentially in the instance of HR, bystander B cell activation resulting from other sexually transmitted infections, and unproductive exposures to HIV such as with individuals with on PrEP, and subsequently more heterogenous than the naïve pre-immune compartment. Such heterogeneity and memory bias of the pre-immune repertoire, similar to the consequences of imprinting observed with responses to other viruses (e.g. influenza, dengue), could result in inconsistent HIV vaccine efficacy among HR. We hypothesize that the composition and responsiveness of the gp120 pre-immune B cell compartment is altered in HR and contributes to inconsistent vaccine responses. To address this hypothesis, we will leverage existing longitudinal samples from US-based HR that are or are not using PrEP and control individuals at low-risk for HIV acquisition (LR) obtained through the NIH-funded MWCCS, a prospective Chlamydia trachomatis infection cohort, and South Africa-based HR and LR that participated in the HVTN 702 vaccine efficacy trial to complete the following aims 1) determine the molecular and phenotypic features of pre-immune gp120+ B cells, 2) define the mobilization of pre-immune gp120+ B cells following vaccination, and 3) identify drivers of pre-immune gp120+ memory B cell development.

NSF Awards · FY 2024 · 2024-08

Highly accurate copying of DNA into RNA is essential for cell growth and proliferation across all domains of life. This project will fill a fundamental gap in our understanding of the chemical reactions involved in copying the information contained within DNA into RNA with a low occurrence of errors. The project will produce a detailed description of how the enzymes involved extend the RNA chain and perform error correction. The results produced will aid the broader scientific community in understanding why higher organisms have evolved three specialized enzymes to copy specific classes of genes when lower organisms use a single enzyme to copy all genes. The project will train students and produce Ph.D.-level experts who will be sought by both the public and private sector for their ability to apply rigorous biophysical approaches. Undergraduate students will also be trained in these approaches. This exposure will allow the students to be more competitive for future careers in STEM. High-fidelity (low probability of error) gene expression is essential for organisms in all domains of life. Archaea and Bacteria employ a single RNA polymerase, RNAP, to transcribe all genes in the prokaryotic cell. In contrast, eukaryotes have at least three RNA polymerases (Pols). Pol I synthesizes ribosomal (r) RNA, Pol II synthesizes messenger RNA and most regulatory RNA, and Pol III synthesizes the 5 S rRNA and transfer RNA. It has been hypothesized that Pol II evolved from the archaeal RNAP and Pol I and III evolved from Pol II via sequence divergence of core subunits and incorporation of transcription factors as bona fide subunits. These observations suggest that the eukaryotic polymerases have evolved divergent mechanisms of transcription and transcription fidelity making them uniquely suited to transcribe their target genes. The project will interrogate the role of subunits within these multi-subunit RNA polymerases that are related to error correction. The overall goal of this work is to employ transient state kinetic approaches to inform the evolutionary divergence by quantitatively defining the kinetic mechanisms of transcription and transcription fidelity for the three Pols. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY People with vision impairments or blindness routinely face substantial challenges in everyday living that can significantly limit their participation in physical activity and exercise important for managing weight and overall health. While there is a growing amount of scientific literature documenting physical inactivity and increased time spent in sedentary behaviors among people with vision impairments, there remains a dearth of health promotion research specifically studying this vulnerable population in comparison people with adequate sight. Further lacking is the availability of evidence-based physical activity-based health promotion programs tailored to the unique challenges experienced by individuals with a vision impairment. Our preliminary findings clearly support the significance of this health disparity within this vulnerable population. As such, this timely project will address this health disparity by: (1) adapting an evidence-based physical activity behavior change intervention tailored to the unique challenges of adults living with a vision impairment and (2) establishing feasibility, patient satisfaction, and preliminary efficacy of the adapted intervention for increasing physical activity intensity and duration along with other health outcomes among adults with vision impairments. Our research will also be informed by an Advisory Board consisting of key stakeholders (e.g., adults living with a vision impairment; vision-related organizations involved in advocacy, policy, and service; along with our investigative scientific team).

NIH Research Projects · FY 2026 · 2024-08

Project Summary: The strategy of selective killing cancer cells while sparing healthy cells holds great promise for cancer therapy, but its effective implementation remains a formidable challenge for numerous cancer types. Targeted therapies have so far primarily focused on inhibiting or modulating the activity of proteins that help cancer cells survive or proliferate, rather than directly targeting the mutations in driver genes. This proposal seeks to bridge this critical void by introducing and evaluating a novel approach. Its primary goal is to assess a method harnessing engineered catalytic RNA molecules, known as ribozymes, for the specific killing of cells expressing mRNA of cancer genes with certain cancer driver mutations. In addition, it will test our recently developed combinatorial method (RABADOCS) for optimizing ribozyme sequences by sampling millions of variants to identify the most specific and sufficiently efficient ones. We will use group I intron ribozymes from the species Tetrahymena thermophila that can be re-engineered to specifically recognize a splice site on a targeted mRNA and replace the 3'-portion of that RNA with a sequence provided by the ribozyme. These ribozymes require a U (uridine) at the target’s splice site and are inactive when this U is absent. This will allow the ribozymes to differentiate between mutant and wild type target mRNA and ultimately between cancer and healthy cells. Our ribozymes will mediate cell killing by replacing the 3'-portion of the target mRNA with a sequence encoding a cytotoxic peptide in-frame. Translation of spliced mRNAs will then induce death for cells expressing the mutant mRNA. As a proof of concept, we will develop and optimize trans-splicing ribozymes that kill cells containing the JAK2 mutation V617F (c.1849G>T), which is the most common driver mutation in classical myeloproliferative neoplasms (MPNs). Aim 1 will optimize the ribozyme’s trans-splicing efficiency and specificity independent of any toxin sequence. The ribozyme’s external guide sequence will be optimized by employing ribozyme libraries and selecting the best variants using RABADOCS. In parallel, aim 2 will test several toxin peptides that are translated from constructs with the same sequence as ribozyme splice products. The results will identify ribozyme-mediated toxicities that maximize the desired killing while minimizing off-target killing. Lastly, in Aim 3 the most promising ribozymes and toxin sequences will be tested in murine hematopoietic stem-cell like cells (HPC-7) expressing wild-type JAK2 and/or JAK2V617F. We hypothesize that our combinatorial approach will yield ribozymes with high target sensitivity and specificity, providing the proof of concept that ribozyme-mediated targeted killing of cancer cells can be achieved. As a by- product, our study may provide the groundwork for a novel therapeutic strategy for JAK2V617F-driven MPNs.

NIH Research Projects · FY 2025 · 2024-07

Abstract Tooth and alveolar bone health are essential for proper oral functions of feeding, chewing, speaking, and other essential activities of daily life. Alveolar bone loss due to periodontitis and tooth cracking are the second and third most common reasons for tooth loss. Elucidating the molecular regulation of dentin synthesis and alveolar osteogenesis will contribute to filling the significant knowledge gap and finding new molecules to target for therapeutics. The runt related transcription factor 2 (Runx2) is the master regulator of skeletogenesis. Humans harboring mutations in the RUNX2 gene exhibit cleidocranial dysplasia, which is characterized by both dental and various skeletal anomalies. Global deletion of Runx2 gene in mice results in complete failure of ossification and embryonic lethality. Our previous work has demonstrated that Runx2 is required for the differentiation of the resting chondrocytes to hypertrophic chondrocytes and endochondral ossification. We have also shown that Runx2 gene deletion in developing osteoblasts impairs the postnatal synthesis of long bone and the acquisition of adult bone mass. Our preliminary data show a surprisingly significant increase in dentin synthesis in mice with Runx2-deficient odontoblasts and a concomitant decrease in bone synthesis and bone mass by Runx2-deficient osteoblasts. Furthermore, the microenvironments of alveolar osteoblasts compared to long bone osteoblasts are fundamentally different. These osteoblasts have different origins, pathogen exposure, and loading forces. Therefore, we hypothesize that Runx2 is required for postnatal dentin synthesis and alveolar osteogenesis. We further postulate that the gene expression profiles of alveolar and long bone osteoblasts are fundamentally different. We will experimentally test these hypotheses in the following proposed aims: In Aim 1, we plan to elucidate the regulatory role of Runx2 in committed odontoblasts for dentin synthesis. Aim 2 is focused on determining the critical differences in Runx2 regulated alveolar and long bone osteogenesis. The proposed experiments will utilize unique mouse models to answer fundamental mechanistic questions about the differential regulation of dentin and alveolar bone synthesis by Runx2. This application builds on a strong interdisciplinary scientific environment and institutional support to train successful clinician-scientists in the field of oral and maxillofacial regeneration. This combination is necessary for my goals of becoming an independent researcher as an oral and maxillofacial surgeon-scientist. The findings from the proposed experiments will uncover Runx2-regulated pathways that could serve as therapeutic targets for dentin and alveolar bone repair and regeneration. Additionally, understanding the functional differences between alveolar and long bone osteoblasts will enhance craniofacial and orthopedic therapies.

NIH Research Projects · FY 2025 · 2024-07

Abstract (30 line max) Diabetes mellitus is the leading cause of kidney failure, heart disease, and stroke, affecting 537 million people worldwide. Diabetes is diagnosed as an increase in blood glucose due to insufficient or ineffective insulin production by the pancreatic β-cell. The inability of the β-cell to adapt to changes in physiologic demand results in hyperglycemia. Under select conditions, such as injury and high-fat diet, the β-cell is capable of expansion, increasing in size and/or number to increase insulin production. Therefore, one potential strategy for mitigating hyperglycemic events is to stimulate β-cell replication. A crucial component for β-cell maturation and β-cell expansion in the adult pancreas is macrophages which support tissue growth through the production of cytokines and stimulatory factors. In models of injury, macrophages shift from their basal classically activated proinflammatory state to a more alternatively-activated anti-inflammatory state, supportive of tissue remodeling through the production of anti-inflammatory cytokines, such as TGF-β. Robust β-cell proliferation has also been measured during pregnancy as a result of reductions in insulin sensitivity and increased fetal demand, making pregnancy an ideal model for investigating expansion in mature β-cells under non-pathologic conditions. β-cell expansion during gestation and macrophage contribution to maternal β-cell growth has not been fully elucidated. I will assess changes in macrophage quantity and phenotype throughout pregnancy. I will determine the source of macrophage accumulation within the islet and whether it is a critical component for β-cell expansion in late gestation. I will also determine whether depletion of islet resident macrophages impairs β-cell expansion in late gestation. I hypothesize that pregnancy drives islet macrophages to an alternatively-activated phenotype, necessary for maternal β-cell expansion. To test this hypothesis, I will explore the following two aims: (1) Characterize the macrophage population within the islet throughout gestation. (2) Establish the requirement of macrophages for maternal β-cell expansion in late gestation. The completion of these aims will identify macrophage-specific responses during pregnancy and their effects on β-cell expansion. 1

NIH Research Projects · FY 2024 · 2024-07

ABSTRACT This proposal requests NHLBI support for the 2024 Scientific Sessions meeting presented by the Council on Basic Cardiovascular Sciences (BCVS) of the American Heart Association (AHA). This meeting has become the “go to” meeting for basic and translational cardiovascular sciences by providing opportunities for established and emerging investigators to present their work and receive constructive feedback. The meeting in 2024 represents the 19th consecutive annual conference. Funding for BCVS Scientific Sessions from National Heart, Lung and Blood Institute (NHLBI) has been secured for 15 of the last 17 years. The BCVS summer conference is entitled “Advances in Cardiovascular Science: From Discovery to Translation”. In 2023, our in-person conference was a rousing success with more than 1,000 investigators from around the world. We plan to again hold an in- person meeting in 2024 to help facilitate the careers and networking opportunities for young investigators, and support collaborations. The meeting is scheduled for July 22-July 25, 2024 and will begin on a Monday and run through Thursday. The conference will highlight the newest basic and translational cardiovascular research with implications for cardiovascular health and disease. There will be approximately 14 scheduled state-of-the-art sessions that include a mix of established and emerging investigators. The 2024 BCVS keynote address will be given by Dr. E. Dale Abel, the William S. Adams Distinguished Professor of Medicine and Chair and Executive Medical Director of the Department of Medicine, David Geffen UCLA School of Medicine. There are specific sessions for early career scientists, including two early career sessions, an Early Career Keynote Lecture, and the Outstanding Early Career Investigator Award Competition. We will host special sessions for networking including interactions with journal editors and staff. We will also include the Women in Science Breakfast to support female scientists. At every step, we will focus on diversity and inclusivity to promote the very best cardiovascular science. This R13 proposal is designed to provide support to young investigators to enable their participation in the meeting as presenters either in oral or poster format. The primary organizers are Drs. Sumanth Prabhu and Farah Sheikh. The Program Committee (11 members) includes the past program co- chair and a diverse leadership from across the US drawn from the many disciplines encompassed by BCVS. The AHA will continue to provide its outstanding administrative support with dedicated staff for the logistics of the conference. We fully anticipate that this team will coordinate an exceptional 2023 BCVS conference.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY/ABSTRACT HIV is declining in many parts of the world, but rising in Latin America, including Brazil. From 2010 to 2019, the number of new HIV infections in Latin America increased 21%, compared to a global reduction of 23%. The number of reported HIV cases in Brazil increased from 13,719 in 2011 to 40,880 in 2021, with increases driven by youth aged 15-24 years. Among men aged 15-24 years, HIV incidence per 100,000 increased from 37.8 in 2000 to 66.5 in 2018 – a 75% increase. HIV pre-exposure prophylaxis (PrEP) was approved in Brazil in 2021 for adolescents aged 15-17 years, but PrEP uptake has been <1% among youth. Our team conducted the first and largest PrEP demonstration study among adolescents in Latin America (PrEP1519), in which we implemented peer navigation strategies that linked 1174 youth (20% of 5870 recruited) to PrEP services at health facilities. In qualitative interviews, youth articulated positive relationships with peer navigators but reluctance to engage with facility-based services due to past experiences with stigma and logistical and structural challenges accessing care. These data suggest that programs featuring peers were welcomed, but alternatives to facility-based services are urgently needed to scale up PrEP among youth. We hypothesize that equipping peer lay workers – trained youth who are integrated into health care teams – to deliver PrEP in community venues will increase PrEP uptake among youth. The goal of this project is to conduct a series of studies to refine, test, and understand experiences with a peer-led community-based intervention (CommunityPrEP, “COMPrEP”). In Aim 1, we will conduct qualitative interviews guided by the Consolidated Framework for Implementation Research to identify key modifications for the PrEP1519 peer navigator intervention to adapt it for community delivery by peer lay workers. In Aim 2, we will randomize 1400 youth who can benefit from PrEP to access PrEP through peer lay workers at community venues (COMPrEP intervention) or health providers at health facilities (standard care). We will conduct intent-to-treat analyses with modified Poisson regression to compare PrEP uptake (primary outcome) across randomized arms. We will follow the subset that initiates PrEP (expected to be N~385) for 12 months to compare secondary outcomes (1-month adherence, 3-month and 12-month persistence). Throughout the trial, we will monitor participant safety through our study team and an external monitoring board. In Aim 3, we will use the RE-AIM framework to explore experiences with the COMPrEP intervention through qualitative interviews with youth, peer lay workers and health providers, and local and national policymakers to develop next steps. The proposed work will provide robust evidence about whether PrEP delivery facilitated by peer lay workers in community settings increases PrEP uptake, adherence, and persistence among youth in Brazil. With members of the National HIV Program in Brazil as our collaborators, we will be well-positioned to provide results to guide programming for this population and scale up the intervention if effective.

NIH Research Projects · FY 2025 · 2024-07

Bacterial infections pose one of the most significant global public health challenges, exacerbated by the emergence of antibiotic-resistant strains. As a result, treating these infections has become increasingly difficult. Recently, the FDA approved cefiderocol, a siderophore-antibiotic conjugate, as a new treatment for urinary tract infections (UTIs) caused by antibiotic-resistant Gram-negative bacteria. This approval highlights the potential of targeting the bacterial siderophore-mediated metal uptake process for pharmaceutical interventions. A crucial step in this process involves the uptake of metal-chelated siderophores across the bacterial inner membrane, facilitated by ATP-binding cassette (ABC) importers. Unfortunately, the detailed molecular mechanisms underlying these importers remains poorly understood, impeding the development of targeted drugs. This proposal focuses on a unique example of these critical importers: the yersiniabactin importer YbtPQ from uropathogenic E. coli, the leading cause of UTIs. Our preliminary studies have revealed that YbtPQ exhibits folding characteristics similar to an ABC exporter, representing a novel type of ABC transporter. Building upon this discovery, we expect to study the fundamental structure-function relationship of YbtPQ by pursuing two specific aims: 1) Define the substrate selectivity and inhibitory mechanism of YbtPQ; 2) Understand the unique structural motifs within YbtPQ. To accomplish these goals, we will employ a multidisciplinary approach, combining mutagenesis, specifically-designed transport assays, microscale thermophoresis, circular dichroism, native mass spectrometry, molecular dynamic simulations, and cryo-electron microscopy. Successful completion of this proposal will yield valuable insights into the substrate/inhibitor selectivity and transport dynamics of YbtPQ. This knowledge will serve as a solid foundation for future drug development targeting this importer, offering new possibilities for effectively treating bacterial infections and addressing the challenges of antibiotic resistance.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Some of the most novel insights in aging have come from studies in model organisms like S. cerevisiae. While a lot has been done about gene (mRNA) expression changes that accompany aging, not much has been done about short RNA changes that may accompany aging. To address this, we sequenced the short RNAome in young and old yeast and discovered that there was a significant induction of a family of short RNAs derived from tRNAs, called tRF-5s or tiR-5s or tRNA halves, that are created by cleavage of tRNAs in the anticodon loop. In addition, we discovered a strong induction of extended transcripts spanning the tRNA loci in the aged yeast compared to the young yeast. In this exploratory project we propose to investigate the tRNA halves and the extended tRNA transcripts to determine if they contribute to lifespan or to the proteostasis that is associated with aging. In Aim 1 we will focus on the tiR-5s, overexpressing or depleting them to study effects on lifespan and proteostasis. We will also determine how the tRNA halves accumulate with age and whether mutations in yeast that advance aging advance the accumulation of the tRNA halves. In Aim 2 we will turn to the extended tRNA transcripts, defining their start and end points, how they accumulate and whether their overexpression contributes to lifespan changes or aging-associated dysregulation of proteostasis. These exploratory studies will define whether these short RNAs and/or the abnormally extended tRNA transcripts contribute to determination of lifespan or to specific disturbances in proteostasis that accompany aging. A positive result will lead to more in depth studies exploring the mechanism by which these tRNA based short and long RNAs are produced and how they contribute to the aging phenotype. This project brings together an expert on yeast aging (Dr. Jeffrey Smith) and an expert on tRNA fragments (Dr. Anindya Dutta) to explore what could be a novel mechanism of aging.

NIH Research Projects · FY 2025 · 2024-07

All organ tissues contain fibroblasts and macrophages. Emerging evidence indicates that direct interaction between fibroblasts and macrophages occur to maintain homeostasis as well as determining disease outcomes, such as asbestos-induced toxicity. The interaction, or communication, between these cells have a role in changing the phenotype of each cell. Activated fibroblasts express sonic hedgehog, and macrophages express proteins in the Hedgehog pathway, including patched, smoothened, and glioma-associated oncogene. Our preliminary data show that macrophages from human subjects have increased expression of Gli1, Smo, and Ptch. Subjects with asbestos-induced toxicity also express significantly more PGC-1a and CPT1A in lung macrophages than normal subjects. Mice harboring a conditional deletion of Shh in fibroblasts blocked Gli1 expression in macrophages, whereas asbestos-exposed WT mice had increased expression of Gli1, PGC-1a, Cpt1a, and increased OXPHOS in macrophages. Recombinant Shh induced expression of acyl carnitines necessary for FAO. These data strongly suggest that fibroblast-macrophage interaction has an important role in asbestos-induced toxicity. We hypothesize that fibroblast-macrophage interactions via Hh signaling mediate macrophage metabolic reprogramming and progressive asbestos-induced toxicity. In Aim 1, we will co-culture human lung fibroblasts (HLF) with resident alveolar macrophages (RAMs) or monocyte-derived macrophages (MDMs) from human subjects to determine which cell subset has Hh signaling. We will also determine mitochondrial bioenergetics to confirm FAO in lung macrophages from subjects with asbestos-induced toxicity with and without silencing Smo. Aim 2 we will determine the fibroblast cell type that is responsible for production of Shh utilizing Cthrc1creER mice. We will also determine the mechanism(s) by which Hh signaling in macrophages mediates metabolic reprogramming to FAO utilizing mice harboring a conditional deletion of Smo in macrophages. In Aim 3, we will determine if constitutive activation of hedgehog signaling in macrophages mediates progression of established asbestos-induced toxicity. We will also determine if disruption of fibroblast- macrophage communication will abrogate this progression. By using innovative techniques, transgenic mice, and human tissue, the studies in this proposal will provide: (a) new insights into fibroblast-macrophage interaction via Hh signaling; (b) an understanding of the molecular mechanism(s) by which fibroblast-macrophage interaction mediate metabolic reprogramming; and (c) proof-of-concept by targeting the Hh pathway genetically and pharmacologically to disrupt fibroblast-macrophage interaction and halt or reverse asbestos-induced toxicity.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Approximately one-third of individuals without dementia at the time of death are found to harbor high levels of Alzheimer’s disease (AD) pathology, including amyloid-β plaques and neurofibrillary tangles, at autopsy. We hypothesize that such individuals exhibit physiological resilience that confers the ability to maintain cognitive function despite the accumulation of AD-related pathologies. The identification of the specific mechanisms by which these older individuals with Alzheimer’s disease pathology avoid dementia is one of the most pivotal, unanswered questions in the field. Cognitive impairment in AD is the result of synapse loss in brain regions that are critical for memory processes. Our work and that of others has demonstrated that synaptic markers and dendritic spine loss correlate more strongly with cognitive impairment in Alzheimer’s disease than accumulation of amyloid-β plaques and neurofibrillary tangles. This implies that the ability to maintain cognitive function in an environment of AD pathology must be linked to the preservation and maintenance of synapses or spines. Aberrant tau accumulation is a strong pathological correlate of cognitive decline both in normal aging and Alzheimer’s disease. Abnormal tau initially accumulates in somatodendritic compartments among layer 2/3 neurons in the entorhinal cortex, and is thought to spread to anatomically connected regions via synaptic connections. These findings suggest that aberrantly phosphorylated tau seeds residing in synaptic compartments are crucial to the spread or propagation of tau pathology in aging and Alzheimer’s disease patients. The extent of neurofibrillary tangle spread through the brain correlates with the severity of cognitive impairment, and thus, halting spread of tau pathology may represent a plausible mechanism of resilience to age- related memory loss or Alzheimer’s disease. This raises important questions: do resilient patients harbor less pathogenic tau seeds in synaptic compartments or are their synapses more resilient to tau? What are the synaptic proteins and cellular pathways that associate with tau seeding and tau-induced synaptotoxicity in resilient patients? The goal of this R21 is to address these provocative questions to open new doors of investigation by identifying putative therapeutic protein targets that are linked to modulating tau seeding, tau- induced synaptic dysfunction and synapse preservation in Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2024-07

Project Summary: We have patented a novel polymer-based in vivo-delivery platform for therapeutic antibodies using a super- hydrophilic polymer, poly 2-methacryloyloxyethyl phosphorylcholine (PMPC) (US20220175857). This platform provides the following features to any antibodies tested: i) prolonged body circulation, ii) specific delivery irrespective of the target locations in the body, iii) protection from the environment and immune surveillance, and iv) selected release at specific locations upon response to the environment. In addition, this platform grants penetrable features to the central nervous system (CNS) and the lymph nodes (LNs), where antibody delivery is greatly challenging. Anti-cancer antibody directing CD20, rituximab, allowed for the complete elimination of systemically metastasized B-cell lymphomas, which are in the CNS and LNs, via engineering with this platform (nRTX). Moreover, animals treated with nRTX evoked a potent anti-cancer immune response through the efficient release of tumor-specific antigens within the LNs. Importantly, this platform has achieved the safe delivery of rituximab in mice, rats, and non-human primates. This year, nRTX has been selected as a collaborator project for the Nanotechnology Characterization Laboratory program toward its clinical translation. Very recently, we successfully simplified this platform for further smooth clinical translation using trastuzumab – a HER2-directing monoclonal antibody – without impairing most features listed above (provisional patent#: 63/579,586). The primary difference in this novel technology from the nanocapsule platform is to ensure the site-directed modification of antibodies by PMPC polymer without masking the epitope recognition capability and Fc-dependent antibody functions, such as antibody-dependent cellular cytotoxicity or Fc-receptor binding. We here applied this new platform to antibody-drug conjugates (ADCs) to improve their anti-cancer efficacy and overcome the known issues surrounding ADCs in clinical use. The site-specific PMPC engineering of ADCs allowed for heightening of the drug-antibody ratio (DAR) by two-fold compared to the parental ADC, Kadcyla (T- DM1), one of the HER2 directing ADCs approved by the FDA in 2013 to treat breast cancers. Moreover, this technology enabled exerting superior anti-cancer activity against various cancerous cells in comparison to Kadcyla, while providing the CNS-deliverable feature. Importantly, PMPC-engineering of Kadcyla with high DAR successfully induced anti-cancer immune responses without changes in adverse toxicity. Under this proposal, we will confirm the effectiveness of the site-directed PMPC-engineering of ADCs against other antigens selectively expressed on human cancer cells derived from different disease backgrounds. We will also test their efficacies in patient-derived xenograft mouse models. At the end, we will develop a novel ADC by further engineering the PMPC polymer structure to overcome therapy-resistant issues of ADC. These studies are intended to develop highly reliable, clinically relevant, and sufficiently safe ADCs.

NIH Research Projects · FY 2025 · 2024-07

Despite the success of antiretroviral therapy (ART), cardiovascular (CVD), liver (LD), and other chronic diseases increasingly replace AIDS-related complications as the most common causes of morbidity and mortality in HIV-1-infected patients. Gut mucosal damage leading to increased intestinal permeability and ongoing chronic microbial translocation is a central factor in persistent systemic immune activation and inflammation and represents a critical driving mechanism of HIV-1 pathogenesis. However, the specific mechanisms and mediators of these processes are not well understood. Neutrophils, the most abundant immune cell population in the body, are specifically geared for sensitive detection of invading microbial and viral pathogens and represent the first and most robust innate immune population responding to microbial translocation. Neutrophil NETosis results in the formation of neutrophil extracellular traps (NETs) that promote endothelial damage, atherosclerosis, and atherothrombosis. NETs provide the stimulus and the scaffold for thrombus formation, prime macrophages for the production of cytokines that amplify immune cell recruitment in atherosclerotic plaques, and induce endothelial damage. We show that neutrophils from HIV-1-infected patients display a high capacity for NETosis and the production of NETs. The goal of this application is to identify the specific subpopulations of neutrophils responsible for NETosis in HIV-1-infected individuals. The central hypothesis of this proposal is that HIV-1 infection is associated with the induction and expansion of specific neutrophilic subpopulations with increased capacity to undergo NETosis. Reactive oxygen species (ROS) and NETs released from activated neutrophils promote organ damage and contribute to the progression of CVD, LD, and other chronic conditions. This hypothesis has been formulated on the basis of our own preliminary data and recently published reports demonstrating the critical role of neutrophils in HIV-1 infection and other chronic inflammatory conditions. The overall objective of the proposed studies is to elucidate the mechanisms responsible for chronic neutrophilic activation and production of NETs in HIV-1 infection in order to reveal the specific checkpoints for intervention. In preliminary studies, we optimized the methods for detailed neutrophil characterization and showed that neutrophils from HIV-1-infected individuals display an activated phenotype, immunosuppressive properties, specific transcriptional profile, increased rate of degranulation, and a high capacity to undergo NETosis. Using a novel method based on cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we identified specific neutrophil subpopulations in chronic inflammatory conditions. Specific properties of the newly identified neutrophil subpopulations strongly indicate that they play a critical role in the pathogenesis of HIV-1 infection. The objectives of this proposal will be accomplished in two Specific aims: 1) Identify neutrophil subpopulations undergoing NETosis in HIV-1-infected individuals, and 2) Determine whether the neutrophil populations undergoing NETosis are expanded in HIV-1-infected individuals and whether the level of NETs in plasma serves as a prognostic marker of vascular and liver disease progression in HIV-1 infection. The significance of the proposed studies is that once the role of neutrophils in the progression of HIV-1 infection is defined, neutrophil activation can be pharmacologically targeted.

NIH Research Projects · FY 2025 · 2024-07

The All of Us Southern Network (AoUSN) has been enrolling in the All of Us Research Program since 2018. It consists of sites in Alabama, Mississippi, and Louisiana, led by a team at the University of Alabama at Birmingham (UAB). In addition to UAB, participating sites include UAB branch campuses in Huntsville, Selma, and Montgomery, AL; Cooper Green Mercy Health Services Authority in Birmingham; University of Alabama in Tuscaloosa; University of South Alabama in Mobile; University of Mississippi Medical Center in Jackson, MS; Louisiana State University Health Sciences Center (LSUHSC) and Tulane University in New Orleans, LA. Two of these sites, UAB, and LSUHSC, are also active in the Nutrition for Precision Health program. The AoUSN was highly successful in enrollments pre-COVID-19, though it took some time for the Network to recover full capacity when enrollments resumed. The AoUSN is currently functioning in an extension phase from July 2023 - February 2024. During this time, it has consistently exceeded recruitment goals, reflecting in part the initiation of multiple innovations that will continue to expand through the funding period of the new OTA. These include: inpatient enrollment at UAB and several collaborating sites; establishment of a new site affiliated with UAB in Dothan, AL; opening “pop-up” enrollment sites throughout the region; use of several mobile clinical research units to reach participants in rural areas; use of Computer-Assisted Telephone Interviews (CATI) to facilitate retention; development of a relationship with Lakeshore Foundation in Birmingham, AL to engage and enroll persons with disabilities. We expect to continue and expand upon these innovations in the next five years. Inpatient enrollment has proved to be especially productive and efficient in terms of staff resources and will be expanded at UAB and at partner sites that have inpatient units. Our experience in setting up a new site in Dothan, AL prepares us to establish additional sites in larger cities in our region. We currently have four mobile units, including one exclusively dedicated to All of Us, and have another on order that will be dedicated to enrollment in rural areas of Alabama, Mississippi, and Louisiana. We have several active pilots intended to improve our ability to retain participants, including use of retention navigators and providing financial incentives for retention. Each of the AoUSN sites is prepared to enroll children (including four with affiliated dedicated children’s hospitals) and will support participant reassessments once these components are launched. In conclusion, the AoUSN remains committed to the principles of the All of Us Research Program to ensure broad inclusion in the program. Precision medicine is a major theme in UAB and our affiliated institutions, and we see All of Us as a key opportunity to engage individuals from our region to ensure that the benefits of precision medicine are widely available in our community.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Alzheimer's disease is a devastating neurodegenerative disease with limited therapeutic options. Progressive accumulation of the pathological markers, hyperphosphorylated Tau (pTau), amyloid-beta, and gliosis begin up to 20 years before cognitive impairment occurs. Published reports from our lab and others have shown that the TgF344-AD rat model displays many of the early pathological abnormalities that occur in patients with AD, including endogenous pTau accumulation in the locus coeruleus prior to other brain regions, Aβ accumulation throughout hippocampus, degeneration of noradrenergic (NA) axons in hippocampus, reactive astrocytes and microglia, as well as a synaptic dysfunction as early as 6 months in the dentate gyrus. Therapeutic strategies that interfere with these pathological changes could slow disease progression. Increasing the post- translational modification, O-GlcNAcylation, has received recent attention, as it competes with serine/threonine phosphorylation on Tau, preventing its hyperphosphorylation and accumulation. Increasing O-GlcNAcylation also increases non-amyloidogenic processing of amyloid precursor protein due to O-GlcNAcylation of gamma secretase. Pharmacological inhibitors of O-GlcNAcase, the enzyme that removes O-GlcNAc moieties from proteins, are currently in clinical trials for the treatment of AD in patients. Using the TgF344-AD rat model and a combination of technical approaches including immunohistochemistry, confocal imaging, Western blot and brain slice electrophysiology, my dissertation research focuses on pharmacologically increasing O- GlcNAcylation in TgF344-AD rats to test whether it is therapeutically beneficial by (1) decreasing pathogenic accumulation of pTau and amyloid-β (Aβ), preventing NA axon degeneration, and decreasing astrocyte and microglia activation, (2) prevents the pathologically increased LTP due to β-ARs function at MPP-DG synapses and (3) maintaining astrocyte function, including astrocytic β-ARs.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Nearly 13,000 new cases of laryngeal cancer are diagnosed in the U.S. annually. Transoral laser microsurgery (TLM) has become the primary surgical approach for early-stage endolaryngeal tumors as TLM provides tumor control while allowing maximal salvage of normal tissue to preserve post-treatment laryngeal function (i.e. voice and swallow). However, no reliable intraoperative approaches are approved to aid the surgeon's subjective visual assessment of tumor borders during TLM, leading to post-TLM recurrence of disease in a substantial proportion of patients. Panitumumab-IRDye800 has been used in multiple clinical trials for near-infrared fluorescence (NIRF) imaging during surgery of various malignancies. Current fluorescence guided surgery (FGS) clinical trials using panitumumab-IRDye800 require intraoperative pathological confirmation of cancer before further surgical actions can be made beyond the standard of care. Marginal biopsies during TLM are limited in volume and number, which confounds pathological assessment and intraoperative attempts to identify clinically occult invasive margins. The lack of FGS clinical studies during TLM in patients with laryngeal cancer has been a barrier in determining if current metrics to guide FGS would be applicable during TLM procedures. Addressing this clinical barrier is a critical step in determining the benefit of intraoperative NIRF to positively impact both TLM surgery and outcomes in patients with laryngeal cancer. We propose a clinical study to demonstrate feasibility for a novel combination of intraoperative optical imaging approaches to detect occult tumor margins in patients with laryngeal cancer. Objective 1 will establish metrics of intraoperative optical imaging that define pathology- confirmed occult laryngeal cancer margins. This objective will be accomplished by correlating intraoperative imaging during total laryngectomy procedures with post-resection tissue analyses including histopathology as the gold standard for true positive and true negative occult disease. Objective 2 will test the accuracy of intraoperative optical imaging metrics to identify occult laryngeal cancer margins during TLM. This objective will be accomplished using imaging metrics to identify positive optical signals beyond the region of treatment planned in patients who undergo TLM. Outcomes from these studies will be used to design a randomized phase 2 trial to test the combined imaging approach to guide TLM in patients with laryngeal cancer. The long-term outcomes of these studies are anticipated to positively impact intraoperative identification of occult malignancy while minimizing loss of critical laryngeal function.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY/ABSTRACT Cancer associated fibroblasts and pancreatic stellate cells (PSC/CAF) and abnormal tumor blood vessels are two major factors that contribute to treatment failure. PSC/CAF contribute to the collagen-rich extracellular matrix (ECM) which impairs drug delivery, promotes cancer cell survival and contributes to an immunosuppressive environment. CAF have been shown to have a similar role in supporting metastatic sites of disease in PDAC. Currently, no agent is available that can simultaneously selectively reduce activated PSC/CAF and normalize angiogenesis. Both activated PSC/CAF and angiogenic endothelial cells selectively express high levels of integrin v3. There is no expression of integrin v3 in normal tissue. We developed ProAgio, an innovative protein drug with a distinct mechanism of action from currently available integrin-targeting agents in targeting integrin v3 at a novel non-ligand binding site. ProAgio induces apoptosis of integrin v3 expressing PSC/CAF and angiogenic endothelial cells with a high efficiency by recruiting and activating caspase-8 at the cytoplasmic domain of. Our preliminary data shows ProAgio has anti-tumor activity in multiple PDAC models and enhances the effect of gemcitabine. Histologic analyses showed ProAgio decreased collagen and depleted PSC/CAF in tumors. In a phase I clinical trial, single agent ProAgio has demonstrated excellent safety profile, promising pharmacokinetic profile and anti-tumor activity in patients with PDAC. We hypothesize that, by inducing apoptosis of integrin v3 expressing cells, ProAgio will specifically deplete both angiogenic endothelial cells and activated PSC/CAF leading to inhibition of collagen deposition in PDAC tumors, which will enhance drug delivery and increase the sensitivity of PDAC to established therapies. This Project has three Specific Aims: 1) To characterize the toxicity and determine the recommended phase II dose of ProAgio in combination with gemcitabine and nab-paclitaxel. We will conduct a phase I clinical trial of ProAgio in metastatic PDAC patients with no prior therapy with gemcitabine and nab-paclitaxel. The goal is to determine the recommended phase II dose, evaluate pharmacokinetics of ProAgio, and characterize toxicity. A secondary objective is to obtain preliminary activity data (overall response rate) for ProAgio with gemcitabine and nab- paclitaxel in PDAC. 2) To analyze the effect of ProAgio in patient tumors and validate the mechanism of drug action. PDAC patients enrolled in the expansion phase will undergo paired pre- and post-treatment biopsies. We will validate mechanism of ProAgio action by assessing changes in intratumoral collagen, blood vessels, PSC/CAF subtypes, immune cells, and αvβ3 expression in these patient samples. Samples will be analyzed via multiplex immunofluorescence with spatial quantification and single cell RNA sequencing. 3) To evaluate the pharmacodynamics effects of ProAgio on angiogenesis and tumor perfusion. The perfusion changes in tumors after treatment with ProAgio plus chemotherapy will be measured using quantitative DCE-MRI in all patients.

NIH Research Projects · FY 2025 · 2024-07

The Center for Clinical and Translational Science (CCTS) Partner Network is situated in the Deep South of the United States; a region that is heavily affected by chronic diseases. The CTSA Predoctoral T32 at the University of Alabama at Birmingham (Deep South Predoc T32) will support the training and advancement of predoctoral PhD and/or MD students in clinical and translational science (CTS) and communication across the translational spectrum from basic scientists to clinical scientists to population scientists from 11 institutions across Alabama, Louisiana, and Mississippi. The mission of the Deep South Predoc T32 program is to inspire and develop future clinical and translational scientists by immersing interdisciplinary trainees in a novel scheme of team science training and peer mentoring to build communication and leadership skills across the translational spectrum. The National Center for Advancing Translational Sciences defines the translational spectrum as representing each stage of research along the path from the biological basis of health and disease to interventions that improve the health of individuals and the public. It is essential to train researchers with skills that match the ever-increasing complexity of the research enterprise with communication skills across the translational spectrum. To address this complexity, we have the need and opportunity to develop translational scientists to go beyond their disciplinary knowledge and have an appreciation for and an understanding of the translational spectrum outside their immediate discipline, enabling effective communication that includes communicating their research to the lay community. Our approach incorporates team science and peer mentoring across the translational spectrum to build CTS research training as well as communication skills in our future leaders. The objectives of the Deep South Predoc T32 program are to: 1) Identify, recruit, and cultivate interdisciplinary medical students and PhD students in health-related, biomedical doctoral programs across the CCTS Partner Network to focus on mentored, translational, rigorous research of chronic diseases prevalent in the Deep South region; 2) Establish and nurture Translational Spectrum teams (TeamS) that incorporate team science and peer mentoring strategies to expand the knowledge and interest in and build communication skills across the translational spectrum; 3) Ensure individualized training in CTS leadership as well as scientific verbal and written communication skills; 4) Promote student well-being with peer and near peer strategies; 5) Provide opportunities for future career options with mini-sabbaticals and inspirational role models; 6) Foster development of expanded networks for mentors, collaborators, and peers to engage within the larger translational scientific community; and, 7) Maintain effectiveness of the T32 programs through evaluations, surveys, and focus groups to implement improvements annually. Our long-term aim is to generate well-equipped clinical and translational scientists who continue to work in teams focused on innovative and creative research.

NIH Research Projects · FY 2025 · 2024-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Elemental processes occurring at the cellular level underpin embryonic development, the function of tissues and organ systems, and disease pathogenesis. The mission of the predoctoral T32 Training Program in Cell, Molecular and Developmental Biology (CMDB) is to provide interdisciplinary training in basic cell, molecular and developmental processes essential for multicellular organisms. While this is a new application for a predoctoral T32 program, it should be noted there is an existing CMDB T32 program at UAB that has been funded by the NIH since 1984. The new CMDB T32 program, which supplants the existing program, capitalizes on training components that have been well-vetted for their effectiveness in providing high quality graduate training. However, these impactful training elements are paired with new training activities designed to meet the ever-changing, and increasingly competitive, scientific environment and workforce. New programmatic activities include didactic courses and workshops focused on basic cell structure, physiology and cell communication, as well as training in cutting edge technologies and scientific ethics. Additionally, a new Career Development Committee has been instituted, which provides CMDB appointees with one-on-one career guidance to prepare trainees for a variety of professional roles. By integrating well-validated training experiences with new activities tailored for the current scientific milieu, the CMDB program will provide trainees with the tools needed to leverage knowledge of seminal cellular processes to elucidate the mechanistic basis of disease. There are 5 components of the CMDB T32 Training program: (1) evidence-informed didactic curriculum and experiential learning activities; (2) research mentoring by outstanding faculty preceptors; (3) training in scientific communication; (4) instruction in responsible conduct of research, scientific rigor and reproducibility; and (5) personalized career guidance and networking opportunities. Trainees will be appointed to the CMDB T32 program in the beginning of their 2nd year of graduate school and support will extend for 2 years. Our goal is to capture students in the early stages of training in order to foster an interdisciplinary mindset as students advance in their research projects. We are requesting 10 predoctoral training positions, based on the large gap between the number of training grant eligible students, and the overall number of predoctoral T32 positions at UAB (an 11th position is supported by matching funds from the UAB School of Medicine). Further support for 10 positions is provided by historical data indicating steep competition for the positions on our existing CMDB T32. Importantly, the CMDB T32 program is unique at UAB in that it is the only T32 program that supports predoctoral trainees in basic cell and molecular biology independent of a disease focus, thus, extending opportunities to high-caliber trainees who would otherwise be ineligible due to the nature of their research. The overarching goal of the CMDB program is to provide a dynamic and multidimensional training experience that equips trainees to translate information garnered from studies of diverse biological systems into biomedical advances that promote human health.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT There is a critical need for researchers dedicated to making improvements in the molecular understanding and treatment of brain tumors to overcome the challenge of therapeutic resistance and better treat these devastating diseases. To accomplish this goal, the University of Alabama at Birmingham (UAB) has developed an innovative Training Program in Brain Tumor Biology to educate highly motivated PhD and MD/PhD students in the fields of fundamental and translational brain tumor research. The requested funding is for 3 pre-doctoral trainees per year in years 1-5 with an additional appointment to be funded by UAB in years 3-5 that will prioritize increasing diversity in neuro-oncology. Each trainee would be appointed for one year with the possibility of an additional year of funding depending on performance. Applicants will be selected at the end of the 2nd year of PhD training with support provided in the 3rd and/or 4th year of graduate school. With the UAB Graduate Biomedical Sciences and the Medical Scientist Training Program as partners, this brain tumor training program builds on the interdisciplinary basic and translational investigative efforts supported by the UAB O’Neal Comprehensive Cancer Center and its Neuro-Oncology Program and the UAB Comprehensive Neuroscience Center. Indeed, 21 faculty members in 11 different departments serve as mentors on this training program. Our faculty are dedicated to training in brain tumor cell signaling, metabolism, drug development, therapeutic resistance, imaging, immunotherapies and clinical experience. Faculty are highly collaborative as documented by co-authored publications, co-funded grants, and co-mentorship of trainees. This committed training environment provides an ideal setting for the implementation of interdisciplinary, translational brain tumor research that synergizes scientific efforts. The Training Program in Brain Tumor Biology provides exceptional educational opportunities in basic and translational research. The coursework encompasses the advanced Special Topics in Brain Tumor Biology Course with world renowned guest speakers as well as the Glial Pathobiology: Special Focus on Brain Tumors Journal Club course. Enrichment activities include the Bioinformatics Experience, Diagnosis and Clinical Management Experience, Imaging Experience, Radiotherapy Experience, Statistics Experience, a monthly Brain Tumor Research in Progress conference, a monthly Glial Biology Works in Progress conference, and the biennial Brain Tumor Training Program Research Retreat and Southeastern Brain Tumor Research Meeting. Collectively, the coursework, research, and enrichment activities will ensure the development of the next generation of basic/translational researchers addressing compelling problems related to brain tumors.

NIH Research Projects · FY 2024 · 2024-07

Women in the United States have a 1 in 8 PROJECT SUMMARY lifetime risk of developing breast cancer. Among the various breast cancer subtypes, triple-negative breast cancer (TNBC) carries the worst prognosis and only 11% TNBC patients with distal metastasis are expected to survive beyond 5 years. Furthermore, current therapies for TNBC also provide marginal and in most cases only short-term clinical benefits. Therefore, novel and more efficacious drugs for TNBC treatment are in urgent need. Most epithelial cells are dependent upon contacts with the extracellular matrix (ECM) for survival and undergo apoptosis when they lose contact with the ECM, a process termed anoikis. However, tumor cells, upon detachment from the ECM, are capable of evading anoikis. The acquisition of anoikis resistance is a critical step that contributes prominently to TNBC tumor growth and metastasis. Thus, anoikis inducers represent valuable therapeutic targets for TNBC treatment. However, the molecular drivers of anoikis resistance that can be therapeutically targeted in TNBC remain largely unknown. Protein kinases are excellent drug targets with over 25 drugs targeting kinases are approved by US FDA for treating a wide-variety of cancers in clinic. Therefore, to identify kinases that confer anoikis resistance in TNBC cells, we performed a kinome-wide shRNA screen and identified the PDZ Binding Kinase (PBK) as a driver of anoikis resistance in TNBC cells. We found that PBK was overexpressed in TNBC and predicted poor prognosis. Furthermore, genetic or pharmacological inhibition of PBK induced anoikis in TNBC cells. Based on these results, we hypothesize that PBK confers anoikis resistance to drive TNBC tumor growth and metastasis. The overall objective is to determine the in vivo role of PBK in TNBC tumor growth and metastasis and evaluate pharmacological targeting of PBK for TNBC therapy. Aim 1 studies will determine the in vivo role of PBK as a driver of TNBC tumor growth and metastasis. First, using mammary fat-pad injection-based orthotopic mouse model of TNBC tumor growth and metastasis we will determine if genetic inhibition of PBK suppresses TNBC tumor growth and metastasis. We will also measure circulating tumor cell (CTC) load in vivo in this mouse model to monitor the effect of PBK inhibition on anoikis induction in vivo. Next, based on our preliminary results, we will test the role of PBK- dependent phosphorylation of transcription factor TWIST1 in reprogramming TNBC cells to acquire mesenchymal cell state and thereby acquiring anoikis resistance. Aim 2 studies will ascertain the efficacy of PBK inhibitor in vivo for TNBC treatment. To do so, we will determine if a highly-potent and efficacious PBK inhibitor, OTS-964 can effectively suppress TNBC tumor growth and metastasis utilizing established TNBC cell lines, and patient-derived xenografts (PDXs)-based models. Taken together, our findings will have strong scientific impact by establishing a novel role of PBK in driving cell state regulatory pathway that facilitate TNBC tumor growth and metastasis as well as by establishing PBK targeting as an effective approach for treating TNBC.

NIH Research Projects · FY 2026 · 2024-07

Under steady-state conditions, >90% of tryptophan (Trp) catabolism occurs in the liver via the kynurenine (Kyn) pathway. However, this process has important pathophysiological roles in the brain, kidneys and heart, as well as in modulating immunity, tumor microenvironments, and pregnancy. Altered Kyn metabolism is observed in aging, cardiovascular disease, organ injury, cancer, and transplantation, where it often is a strong predictor of outcome. Despite their relevance, the mechanisms behind the effects resulting from changes in systemic and local Kyn metabolism are poorly defined. Using untargeted and targeted mass spectrometry together with isotopically labeled standards, we discovered the endogenous formation of the electrophilic mediator kynurenine- carboxyketoalkene (Kyn-CKA) secondary to in vivo Kyn deamination in humans and mice. Kyn-CKA reacts covalently with cysteine residues in transcription factors, transcriptional regulatory proteins and enzymes to regulate protein and cellular function. We recently published that Kyn-CKA modulates TLR/NF-κB- and Keap1/Nrf2-dependent signaling and our preliminary data shows that Kyn-CKA also affects bioenergetic metabolism and impacts seemingly cysteine-independent processes. Furthermore, we now know that N-formyl- kynurenine (NFK) and 3-hydroxy-kynurenine (OH-Kyn) also generate electrophiles in vivo. We hypothesize that Trp catabolism via the Kyn pathway results in the formation of novel bioactive products that adaptively modulate cellular responses via redox-dependent mechanisms. We propose to characterize the mechanisms that regulate the formation of Trp-derived electrophiles via modulation of individual enzymes in the Kyn pathway and the identification of potential enzymes capable of catalyzing NFK, Kyn and OH-Kyn deamination. Trp-derived electrophiles undergo conjugation to glutathione and reductive inactivation, thus we will identify and kinetically characterize the enzymes involved in these processes. Unbiased high resolution mass spectrometry and isotopically labeled precursors will be used to discover pathways for inactivation and biotransformation of Trp- derived electrophiles. Furthermore, we will characterize the effects of Trp-derived electrophiles on cellular and tissue bioenergetic metabolism, first using hepatocytes and then expanding to other cell types as informed by in vivo formation data. In this regard, we will establish the impact of dietary Trp intake regulation on systemic and tissue Trp-derived electrophile levels, as well as assess a potential contribution of the microbiome to this process. Finally, we will use spatial transcriptomics in conjunction with click chemistry-dependent labeling strategies to elucidate the sites of azido-Kyn deamination in vivo and define the impact of this process on electrophile-targeted and untargeted regions of interest to differentiate between proximal and paracrine signaling effects. Overall, our discovery that the Kyn pathway is a source of bioactive electrophiles has the potential to transform our understanding of Trp biology. We expect our research provide a solid biochemical foundation that will enable the development of novel clinical approaches for conditions in which Trp metabolism is dysregulated.

NIH Research Projects · FY 2026 · 2024-06

ABSTRACT There is a fundamental gap in our current understanding of how Mycobacterium tuberculosis (Mtb) causes disease in the human lung, and several reasons are responsible for this lack of knowledge. Firstly, the availability of tuberculous human lung tissues plummeted with the introduction of antibiotics in the 1940s and 1950s. Secondly, TB research using animals rapidly progressed and novel technologies were applied to these model systems. However, it is well known that no animal model reproduces the full spectrum of disease as it occurs in humans, or naturally mediates transmission to new hosts. Not surprisingly, interest in human pulmonary TB was revived decades later when it was realized that the disease had never been eradicated. Despite this, there are few studies focused on elucidating the fundamental mechanisms of active, subclinical, and latent human TB, which is likely due to limited access to human TB lung tissue. We believe the TB field can no longer rely on animal models that yield findings of limited clinical relevance. Thus, there is an urgent need to better understand human pulmonary TB in order to develop clinically relevant therapeutic strategies. The overall goal of this R24 proposal is to transform the global landscape of TB research by accelerating the study of human TB tissue. We will accomplish this by providing user-requested services comprised of two components: (i) the pathological, cellular, structural, and/or genetic analysis of resected human TB or postmortem (PM) tissue and (ii), the dissemination of tissue to the global TB research community. We have several unique advantages since AHRI, located in Durban, South Africa, has access to a continuously growing collection of active TB/HIV and PM samples, including entire lungs/lobes. This R24 proposal is built upon substantial published data from our group that clearly demonstrate our capacity to examine resected and PM lung tissue from Mtb/HIV-infected human subjects. The rationale for this proposal are (i) that by providing this dedicated service to the TB research community, numerous laboratories worldwide will be able to rapidly ascertain the clinical relevance of their basic science discoveries, which will help refine the focus of their research to have translational impact, and (ii) our services will drive the discovery of new human TB paradigms, or challenge existing paradigms, ultimately leading to the development of clinically relevant diagnostics, anti-TB drugs, and/or vaccines. This proposal is significant because it is the first step in a progression of innovative services to the global TB research community that is expected to bolster and accelerate translational and clinical research to expedite the discovery of innovative new strategies to improve diagnosis, prevention, and treatment of TB.

NIH Research Projects · FY 2025 · 2024-06

ABSTRACT Traumatic brain injury (TBI) increases the risks for cognitive impairment and dementia, such as Alzheimer’s disease and Alzheimer’s disease-related dementias (AD/ADRD). Multiple human imaging studies of the brains after traumatic brain injury reported the signs of prominent microglial activation in the thalamus that highly correlates with cognitive impairment. Nevertheless, the role of thalamic microglia in cognitive impairment after TBI remains unclear. We have recently discovered a unique role of thalamic microglia in cortical injury-induced recognition memory deficits in mice. Our preliminary studies demonstrated that local depletion of thalamic microglia, not hippocampal microglia, counteracted recognition memory deficits. Thalamic microglial depletion also improved thalamic neuronal survival and restored c-Fos expression in multiple recognition memory-related brain regions, including the medial prefrontal cortex (mPFC), hippocampus (HPC), and perirhinal cortex (PRh). Local activation of thalamic neurons restored recognition memory deficits, highlighting a primary role of thalamic neuropathology in the behavioral phenotype. Our single-cell RNA sequencing (scRNA-seq) data revealed the development of microglial subtypes in the thalamus, including the one with high phagocytic capability. Consistent with the molecular findings, the thalamic microglia in the injured brains engulfed neurons and inhibition of phagocytosis attenuated recognition memory deficits in the injured mice. These data suggest that thalamic microglia may promote recognition memory deficits by damaging neurons and synapses. Thus, in the proposed study, we will test our hypothesis that excessive microglial phagocytosis in the thalamus after cortical injuries damages neurons and synapses, causing cognitive impairment. We will use a well-established cortical controlled impact (CCI) model in mice. In Aim 1, we will elucidate cortical injury-induced microglial changes and their effects on neurons in the thalamus. In Aim 2, we will demonstrate that removal of thalamic microglia has an immediate restorative effect and a long-term ameliorative effect on recognition memory in the injured mice. In Aim 3, we will evaluate the effects of microglial phagocytosis inhibition on thalamic neuronal pathology and cognitive impairment after cortical injuries. Together, this study will assess the role of thalamic microglia phagocytosis in cognitive impairment after cortical injuries. The findings will provide a novel mechanistic insight into the critical thalamic pathology underlying TBI-induced long-term neurological deficits.