Washington University

NSF Awards · FY 2024 · 2024-08

This award supports a research project in noncommutative geometry and its applications. Noncommutative geometry is a mathematical field in which spaces are studied through an algebraic lens, in a way analogous to the relationship between a topological space and the algebra of continuous, complex-valued functions on that space. An important feature of the algebraic structures in noncommutative geometry is that – unlike multiplication of functions – their multiplication is not commutative, that is, the order in which elements are multiplied changes the outcome. This property makes for a mathematically rich generalization of classical geometry and enables connections between noncommutative geometry and many other fields, including representation theory, differential geometry, and algebraic topology, as well as the mathematical foundations of quantum mechanics. This project will explore the noncommutative geometry of two systems of non-commuting operators, one arising from the theory of Toeplitz operators, and the other from group symmetries. The PI’s primary aim is to introduce new numerical invariants to distinguish noncommutative spaces, and refine known invariants such as the Connes-Chern character. The project will also provide research opportunities for graduate students and postdoctoral scholars and support the PI’s outreach efforts to middle school and high school students in the community. Cyclic theory, the noncommutative analogue of de Rham theory in differential geometry, will be applied in this project to study an analytic version of the Milnor fibration, a novel concept arising in the investigation of the Arveson-Douglas conjecture. A new method for analyzing variations of traces and cyclic cocycles on the fibration will be developed, building an analytic approach to secondary invariants. Additionally, a geometric model for the cyclic cohomology of a Lie group will be constructed to study invariants for proper cocompact actions. This model will facilitate the exploration of new homotopy invariants associated with diffeomorphism groups. 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

SUMMARY Since its inception in 1985, the goals of the Kern Lipid Conference have been to promote metabolic science and increase understanding of metabolic pathobiology and cardiovascular disease. The conference strives to provide an inclusive, open forum for established and young emerging scientists from academia and industry. The 2024 Kern Lipid Conference will be co-chaired by Drs. Jonathan R. Brestoff, Nada A. Abumrad, and Gregory Steinberg and will have the theme “Lipids and Mitochondria in Cardiometabolic Diseases.” It will be held August 12- 14, 2024 at the Viceroy Hotel in Snowmass, Colorado. The primary goals of the 2024 conference are (1) to provide a dynamic interactive environment that leads to novel insights into disease mechanisms involving lipid metabolism and mitochondrial function and (2) to engage and inspire young scientists from diverse backgrounds to develop their careers studying lipid biology, mitochondria metabolism, and cardiometabolic disease pathogenesis. We will examine multiple organ systems including the cardiovascular system, heart, liver, brain, and adipose tissue while delving into metabolic diseases including obesity, diabetes, nonalcoholic fatty liver disease, and atherosclerosis, and other associated conditions such as cancer and neurodegeneration. The program will feature 5 sessions on exciting new areas of research: (1) Lipids and Mitochondrial Metabolism in the Brain, (2) Immune cells and Lipids in Cardiometabolic Diseases, (3) Mitochondria Transfer in Cardiometabolic Diseases, (4) Lipids and Mitochondrial Metabolism in the Regulation of Cardiometabolic Disease Pathogenesis, and (5) Lipids in Cancer. There will be 33 talks over a 3-day period (10 from early career scientists), an evening poster session, and career development sessions for early career scientists. Breaks, lunches, a closing dinner, and a closing reception on the premises promote interactions between participants. Idea exchange is maximized by allocating ample time for questions and discussion after oral presentations. The Kern Conference has traditionally attracted many early career scientists (~40% of the participants) and established scholars in their fields as well as creative scientists from industry. Participation of young scientists is strongly promoted with oral presentation opportunities, the poster session, inviting them to serve as session chairs, and awarding 3 prestigious early-career awards that will advance their career development: the Roger Davis Investigator Award for Transitional Faculty, the David L Williams Lecture and Scholarship Award, and the Franz Simon Poster Award. The setting and meeting format will facilitate interaction and discussion among attendees, making it likely that novel insights into disease mechanisms and therapeutic approaches for cardiometabolic diseases and associated comorbidities will emerge from the 2024 Conference. This R13 application is critical to the success of the 2024 Kern Conference because it will enable us to support the attendance and highlight the research from more early career scientists (graduate students to Assistant Professor), especially those who are from underrepresented groups in biomedical research.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Chronic pain is a leading cause of disability worldwide, drastically decreases quality of life, and can drive the development and maintenance of both alcohol (AUD) and opioid (OUD) use disorders. Both alcohol and opioid use are reported for pain relief, and co-morbid use of both substances is increasingly common, increasing the negative effects of either substance used in isolation. Currently, there is limited research, preclinical or clinical, regarding co-occurring alcohol and opioid use is the context of chronic pain. The kappa opioid receptor (KOR) and its endogenous ligand dynorphin (DYN) is an endogenous opioid system that is widely expressed throughout the brain and heavily implicated in alcohol drinking, opioid seeking, and pain chronification. Our lab has recently identified a novel circuit of nucleus accumbens (NAc) projecting dynorphinergic central amygdala (CeA) neurons that is modulated by persistent pain and contributes to negative affect. However, this circuit has not been investigated in alcohol or opioid use. Here, I propose to test the role of DYN CeA-NAc signaling in 1) binge-like alcohol drinking during persistent pain and protracted fentanyl abstinence, 2) pain avoidance-like behavior and the effects of alcohol on this behavior, and 3) the effects of binge alcohol on reinstatement of fentanyl seeking behavior. I will use a combination of behavioral, photometric, and chemogenetic techniques to test the overarching hypothesis that persistent pain and fentanyl abstinence inhibit a novel dynorphinergic CeA-NAc circuit to drive binge-like alcohol drinking and subsequent effects on cognitive/motivational pain behaviors and reinstatement of fentanyl-seeking in a sex-dependent manner. The proposed work will fill a gap in our knowledge regarding polysubstance use in the context of chronic pain and provide valuable training to a promising young alcohol neuroscientist.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY / ABSTRACT Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can colonize the bladder (cystitis) and ascend the ureter leading to infection within the kidney (pyelonephritis). Though less frequent than cystitis, pyelonephritis carries increased health risks, including hypertension, renal abscess formation, and renal scarring. Nevertheless, the mechanisms of UPEC pathogenesis during pyelonephritis have remained elusive due to lack of an optimal preclinical infection model. Published work from our group has established that males exhibit an elevated susceptibility to chronic pyelonephritis and renal abscess formation. This inherent sex bias in UTI outcomes is androgen dependent, leading us to create new published models of ascending UTI in androgen-exposed female mice that enable investigation of UPEC virulence strategies within the kidney. The UPEC secreted toxin alpha-hemolysin (HlyA) elicits cell lysis and ATP release from various mammalian cell types in vitro, but was previously shown to be dispensable during UPEC cystitis. Preliminary studies using our updated mouse model of ascending UTI demonstrated that HlyA is required for optimal UPEC colonization and associated with severe pathology and renal abscess formation in androgen-exposed hosts. This initial work is congruent with epidemiologic data showing that ~80% of clinical UPEC isolated from patients with pyelonephritis are HlyA positive, versus only 40% of cystitis isolates. Purinoreceptors (P2X) are host cell membrane channels that open upon sensing extracellular ATP and facilitate non-selective passage of small cations. Interestingly, the P2X4 isotype expressed ubiquitously throughout the renal tubular epithelium, is allosterically activated by direct interactions with testosterone; modulation of P2X4 function by testosterone increases receptor affinity for ATP, driving increased ion permeability and subsequent pore conductance. We have found that P2X receptors amplify HlyA cytotoxicity in vitro. Specifically, exogenous testosterone significantly enhances HlyA-mediated lysis of renal epithelial cells, and this effect is abolished in the presence of a non-selective P2X inhibitor. Leveraging updated models of ascending UTI, small-molecule P2X inhibitors, and our in vitro model of renal collecting duct infection, we will test the central hypothesis that UPEC alpha-hemolysin drives renal pathology during pyelonephritis, and its activity is augmented by testosterone via enhancement of P2X channel function. Completion of this work will delineate the contribution of HlyA to UPEC pathogenesis within the kidney, determine the mechanism which facilitates the sex-biased pathogenic effect of HlyA, and define the contribution of pharmacologically targetable host P2X4 channels to HlyA toxicity during pyelonephritis.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Background: Bacteria possess an extensive set of anti-viral immune systems to block the intracellular replication of lytic bacterial viruses (bacteriophages or phages). Among these anti- phage defenses, toxin-antitoxin (TA) systems are common, two-component genetic modules containing a lethal toxin and its cognate neutralizing anti-toxin. TA systems serve as primed defenses: expressed before infection, the antitoxin counteracts its toxin until it senses infection and frees its toxin to block intracellular phage replication and kill the host bacterium. Recently, we identified DarTG1 as an anti-phage TA system (LeRoux 2022). DarTG1 consists of (1) an ADP-ribosylase toxin, DarT1, that modifies single-stranded DNA (ssDNA) to block phage DNA replication & transcription and (2) a glycohydrolase antitoxin, DarG1, that removes ADP- ribosylation during normal bacterial growth. Despite knowing how DarTG1 defends against phage, it is unknown how DarTG1 senses phage infection while avoiding lethal spontaneous activation. Goal/Preliminary Data: As DarT1 is always active when expressed without DarG1, I hypothesize that DarG1 senses phage infection. Thus, I aim to elucidate how DarG1 senses viral infection and stops counteracting DarT1. At present, only two TA systems have known activation mechanisms during phage infection—host transcriptional shutdown and viral capsid expression—and DarG1 senses neither. Instead, my preliminary data suggest that DarG1 senses phage DNA metabolism. First, we identified diverse phage DNA metabolism proteins that activate DarT1 in the absence of infection, indicating that DarG1 senses a DNA-related process. Second, DarG1 physically associates with E. coli’s single-stranded DNA binding protein (SSB) before infection, suggesting that DarG1 localizes with SSB to ssDNA found at DNA replication forks and DNA lesions. Thus, I hypothesize that DarG1 monitors ssDNA metabolism for infection-induced perturbations. Approach: I will determine which phage DNA metabolism processes co-localize with DarG1 during infection and which are necessary for DarG1 to sense infection (aim 1). In parallel, I will identify which DNA metabolism processes are sufficient for DarG1 to react in uninfected E. coli by introducing targeted DNA perturbations & known E. coli DNA metabolism proteins (aim 2). Significance: Uncovering how DarTG1 senses and initiates a response to phage infection will enhance our understanding of anti-viral immunity, as DarG1-like NADAR-containing proteins are found across prokaryotes & eukaryotes, suggesting any DarG1 surveillance mechanism identified may be echoed in other prokaryotic & eukaryotic anti-viral systems.

NIH Research Projects · FY 2024 · 2024-08

Project Summary/Abstract Birth defects occur in 1 in 33 individuals and result in yearly healthcare costs in the United States of America of >$23 billion. Understanding genetic causes of birth defects can provide insights into molecular pathways and ultimately provide a path towards therapeutics. The Gabriella Miller Kids First Pediatric Research Program with its short name of “Kids First” is a program that was created to investigate cancer and birth defects in children. In this program, whole-genome sequencing (WGS) has been generated in combination with collection of phenotype(s). The R03 PAR-23-075 on “Small Research Grants for Analyses of Gabriella Miller Kids First Pediatric Research Data” encourages submission of applications focused on analyzing Kids First. In this proposal, we specifically focus on detection and analyses of de novo variants (DNVs) in the cohorts with birth defects. DNVs are important in human diseases and have been shown to contribute to birth defects. We have developed several tools to detect, quality check, and statistically assess DNVs in both the nuclear and mitochondrial genome. The Kids First Data Portal does not currently have DNV callsets from either the nuclear or mitochondrial genomes. DNVs are likely contributors to the birth defects represented in Kids First and it is critical to generate a comprehensive high quality DNV callset for this dataset. Through this R03 mechanism, we will generate nuclear and mitochondrial DNV callsets for all 6,080 families with birth defects in Kids First for use by others and ourselves. Once we have the DNV callsets we will test for enrichment of protein-coding DNVs in relation to birth defects. Protein-coding DNVs are the most straightforward to interpret and we are well prepared to perform these analyses as evidenced by our previous work. The two aims we will pursue in this grant include comprehensive DNV calling and assessment of protein-coding DNVs in the nuclear genome (Aim 1) and the mitochondrial genome (Aim 2). There are three expected outcomes of our study including 1) identification of new genes involved in birth defects, 2) comprehensive DNV callsets for use by others with interest in birth defects, and 3) integration of our DNV tools (HAT, EGP) in the Kids First Data Portal for use as additional families are added to the portal. Overall, our work will make substantial progress in understanding genetic aspects of birth defects.

NIH Research Projects · FY 2025 · 2024-08

Psychiatric disorders affect up to 30% of all youth prior to age 18, can be highly impairing, and up to 50% of affected children remain symptomatic despite the best available treatments. Part of the challenge is that the altered brain development associated with developing a psychiatric disorder may begin at birth or earlier. Thus, interventions may need to be implemented early in infancy, at a time when the brain is most plastic (susceptible to external manipulations). One preventative strategy would be to target interventions to specific brain networks when they are most plastic in high-risk individuals. Before we can enact this vision, however, we need to learn substantially more about mechanisms of plasticity in neonates. The neonatal period is a time of rapid development, with week-to-week brain changes, and individual neonates vary in their brain organization and in their specific stage of development. Thus, examination of mechanisms of plasticity in neonates will require measurement of week-to-week changes and personalized measures of plasticity and brain organization. This proposal tests the hypothesis that high regional spontaneous activity in individual neonates reflects their personalized brain regions that currently have high plasticity and thus are most responsive to interventions. This hypothesis is evaluated by testing whether spontaneous activity is highest in the personalized brain networks known to be rapidly developing during the neonatal period, predicts which brain regions will have the most week-to-week developmental changes in their connections, and predicts which brain regions will have the most change in response to an intervention directed at sensory networks (massage) in individual neonates. We test these hypotheses using precision functional mapping (PFM) in 80 neonates at two timepoints one week apart. Between timepoints, half undergo 45 minutes of daily massage, an established neonatal intervention. Massage is used as a proof-of-concept to study impact on sensory systems when they are highly plastic. PFM collects 1+ hours of magnetic resonance imaging (MRI) over 2 days to derive reliable measures permitting highly powered within-subject analyses. PFM also permits brain activity and brain connections to be measured within personalized networks, which is required for neonates because of high variability in the specific stage of development. Results are expected to characterize mechanisms of plasticity and provide a model for how to target specific interventions to specific brain networks when they are most plastic, optimizing neurodevelopmental trajectories and reducing psychiatric risk. While this proposal uses massage as a model to influence sensory networks when most plastic, this blueprint can be extended to other brain networks and interventions, reducing risk for psychiatric illness. Results could ultimately have high public health impact by highlighting a guide to optimally time and target interventions to prevent psychiatric illnesses in high-risk infants.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb) caused an estimated 1.6 million deaths in 2021. The emergence of drug- resistant strains has exacerbated the epidemic, often rendering existing tuberculosis (TB) therapies inadequate and revealing the dire need for new agents with unique mechanisms of action to combat the epidemic. Towards this end, we have identified a new series of nitro-containing 1,2,4-triazole compounds that are potent inhibitors of Mtb. Our preliminary data show that the nitro 1,2,4-triazoles retain activity in Mtb strains that are resistant to the frontline antibiotics isoniazid and rifampicin, as well as moxifloxacin. In addition, we provide data that argues against the nitro 1,2,4-triazoles inhibiting QcrB and MmpL3, two proteins commonly targeted by compounds identified in phenotypic screens in Mtb. Thus, it is possible that the nitro 1,2,4-triazole series represents a new mechanism of action (MOA) for inhibition of Mtb. To begin to investigate how the nitro-1,2,4-triazoles inhibit Mtb, we selected for resistant mutants and identified resistant isolates with mutations in genes required for coenzyme F420 biosynthesis and the nitroreductase Ddn. These mutants are also resistant to pretomanid, a prodrug that requires activation of its aromatic nitro group by F420-dependent-Ddn activity in order to exert anti-Mtb activity. We predicted that our nitro 1,2,4-triazoles are similarly activated by Ddn. Therefore, in an effort to circumvent the need for activation, we performed structure activity relationship (SAR) analyses and discovered that replacement of the nitro groups in the phenyl ring on the 1,2,4-triazole core with chloro retained activity against WT Mtb and avoided loss of activity in F420 and Ddn mutants. Thus, the chloro-containing 1,2,4-triazoles circumvent the predominant resistance mechanism against aromatic nitro-containing compounds. We also examined core modifications that would retain anti-Mtb activity and discovered that the 1,2,4-triazole core can be replaced with a chiral pyrrolidine core and retain the same activity. This led us to hypothesize that the core is a “spacer” linking the other parts of the molecule. Since the core structure defines the compound class, our SAR method has now resulted in a second new class anti-Mtb agents that will act as a bioisostere with similar steric volume and retained hydrogen-bond donor/acceptor atoms as the 1,2,4-triazole series. The increased sp3 character of the pyrrolidine scaffolds could be advantageous over the 1,2,4-triazole scaffold in terms of a more diverse chemical space that has been shown to translate to enhanced clinical success. Our objectives are to demonstrate preclinical proof-of-concept for the 1,2,4-triazole and pyrrolidine compounds to combat Mtb infection and optimize lead compounds for pharmacologic properties required for translation to a therapeutic. Based on our preliminary data, we hypothesize that the 1,2,4-triazole and pyrrolidine compounds operate through a unique MOA as compared to other antibiotics and, therefore, dissecting their mode of inhibition will also reveal new biological insight into Mtb physiology and pathogenesis.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Inborn errors of immunity (IEI) are genetic disorders of the immune system that can present with a wide diversity of signs and symptoms. Specific antibody deficiency (SAD) is among the most common classes of IEI and occurs when patients have normal levels of serum immunoglobulins but are unable to form specific responses to antigens. Despite its high prevalence, the pathogenesis of SAD remains poorly understood. Here, we investigate a family that presented with a specific antibody deficiency with an autosomal dominant inheritance pattern. Although whole exome sequencing did not reveal any variants in known IEI-associated genes, whole genome sequencing uncovered a heterozygous tandem duplication on chromosome 14 that intersects the immunoglobulin heavy chain (IgH) locus. All affected individuals also had a selective memory (CD27+ CD38 Lo/Int) B cell deficiency in peripheral blood. CD27- B cells were hyperproliferative in response to T-dependent and T-independent stimuli yet showed diminished differentiation to plasmablasts when stimulated with CD40L and IL-21. Intriguingly, one of the duplicated genes, JAG2 was upregulated more than 100-fold at baseline when measured by bulk RNA-seq. CITE-seq revealed that JAG2 is uniquely overexpressed in patient B cells but not in other immune cells. Given the diminished differentiation and B-cell-specific overexpression of JAG2 seen in these patients, we hypothesize that the duplication has transposed JAG2 under the transcriptional control of the IgH locus, a phenomenon known as ‘enhancer hijacking’, resulting in its increased expression only in B cells. We further hypothesize that the sustained expression of JAG2 upon stimulation results in diminished differentiation, leading to the clinical phenotype of SAD. Throughout this project, we will investigate the effects of JAG2 on B cell differentiation and explore the genetic mechanism of JAG2 overexpression using a number of cutting-edge techniques and novel models, including tonsil organoids and measles-pseudotyped lentivirus to manipulate the expression of JAG2 in human B cells and assess its effects on B cell differentiation. Previous studies of IEI have primarily focused on single nucleotide variants causing altered protein function or abundance. Here, we propose a structural variant is causing altered enhancer interactions between JAG2 and the IgH locus, resulting in SAD. This represents a novel genetic mechanism of IEI and would alter the way we approach the genetic diagnosis of IEI.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT DNA encodes all of the information required to generate and maintain each cell within the body. However, DNA alone is not sufficient and requires the genome to be labeled as active or inactive in a process termed epigenetics by modifications to DNA itself or to histones around which all DNA is wrapped. These epigenetic modifications are well established regulators of cellular development and differentiation, and their function is essential for proper retinal development. While modifications to histone tails such as acetylation and methylation have been extensively studied, we have not yet explored the roles of histone variants in the retina. In fact, histone H3 has two isoforms H3.1/2 and H3.3, which are incorporated in a cellular replication dependent and independent manner respectively. Recent evidence has emerged that mutations within H3.3 cause syndromic disorders, which include severe neurodevelopmental issues, including in some cases retina degeneration and vision loss. In this proposal, we aim to investigate the role of H3.3 in retinal cell fate specification and differentiation. We have preliminary evidence that a mouse model lacking H3.3 in the developing eye causes loss of a subset of retinal cell types. Additional data suggests H3.3 deposition is tightly regulated early in development to control expression of an essential subset of genes. Therefore, in Aim 1 we will complete a full phenotypic characterization of retinal development upon loss of H3.3 from retinal progenitors and post-mitotic neurons. In Aim 2, we will investigate the molecular role of H3.3 in the establishment of the mature retinal epigenome. The results of this study will deliver the first in depth investigation of retinal phenotype after depletion of H3.3 and define the role of H3.3 in retinal epigenetic remodeling and homeostasis. A better understanding of the role of H3.3 in the retina is essential to better understand retinal development and for the design of treatment paradigms for patients with H3.3 mutations.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Multiple sclerosis (MS) is an autoimmune disease that affects nearly 3 million people. MS manifests when the immune system attacks myelin, the insulating sheath around nerve fibers, and myelin producing cells in the central nervous system (CNS). As a result, MS patients suffer from motor, sensory and cognitive deficits. The most common form of MS, relapse-remitting MS (RRMS), is characterized by repetitive cycles of disease and recovery. Tragically, if left untreated, two-thirds of RRMS patients progress to secondary progressive MS (SPMS), characterized by continuous disease progression and irreparable CNS damage. To date, the cause of MS is unclear and there is no cure. To identify potential targets for therapeutic interventions, it is critical to define the cellular and molecular mechanisms underlying MS disease progression. Microglia, long-lived CNS immune cells, have been shown to respond to disease by altering their morphology and gene activity. Preliminary data have shown that this acquired disease-associated microglia (DAM) phenotype is responsible for clearing myelin debris to facilitate remyelination, indicating that DAM could be harnessed to protect against MS progression. To understand the cellular programming underlying microglia function in the context of MS, this project aims to both define the gene specific gene signature of DAM, and define how the function of these cells is affected by repeated activation, such as occurs in relapse- remitting MS. By revealing the mechanism and functional impact of microglia reactivation, this proposal will directly address an outstanding question in the field about how long-term activation of microglia may contribute to MS disease progression. Importantly, microglia, and more specifically, the DAM phenotype appears in chronic neurodegenerative diseases. Therefore, understanding the long-term changes of chronically activated microglia in this project can provide new insights for other disease models in which microglia have been implicated. Upon completion of the proposed project aims, this fellowship would have supported my technical and professional development. Specifically, by completing this project, I would have developed independence in designing, executing, and analyzing experiments to understand epigenetic reprograming of microglia, as well as learning to use a novel model system for studying microglia activation. Additionally, I would have developed my oral and written scientific communication skills. Ultimately, support from this fellowship will help me achieve my long-term goal of becoming a tenured faculty member at an R1 institution.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Candidate: Dr. Jennifer Foltz’s doctoral and post-doctoral training has focused on improving natural killer (NK) cell therapy, a type of innate lymphocyte that has shown clinical efficacy in clinical trials of acute myeloid leukemia (AML). Dr. Foltz seeks to integrate her wet-lab and computational training to delineate the mechanisms of resistance to NK cell therapy. Her long-term career goal is to translate these findings into the clinic. Research Career Development Plan: Dr. Foltz is pursuing an independent tenure track investigator position. During this project, she will expand her expertise in genomic research and enhance her skills as a leader and mentor, which is necessary for effectively running a research laboratory. She will attend relevant genomics and epigenomics seminars and journal clubs, participate in early investigator networking groups, leadership courses, grant writing workshops, and present her research at national and international conferences. Research Project: The long-term goal of her proposal is to define novel mechanisms underlying the regulation of NK cell anti-leukemia function and to translate these findings into the clinic through her established collaborations. Recently, NK cell therapies have included NK cells that possess memory—the ability to remember a prior activation, culminating in a more rapid and proficient response upon a secondary challenge. Cytokine-induced memory-like (ML) NK cells are generated through brief IL-12/15/18 activation, followed by differentiation in vitro, or in vivo within AML patients to become ML. Patients treated with ML NK cells have improved clinical outcomes compared to cNK cells; however, not all patients respond. Based upon previous literature and our preliminary data, we hypothesize that ML NK cells exert unique immune pressure on AML driving differential AML resistance mechanisms and unique activation of ML NK versus conventional NK (cNK) cells. This will be interrogated in the following aims: Aim 1: We will elucidate the transcription factors and epigenetic changes underlying cNK versus ML NK cell anti-leukemia functionality. Here we will determine the role of TOX, transcription factor on ML NK cell function, using CRISPR loss-of-function (LoF). We will also define how AML modulates the epigenome of ML and cNK cells with multiomic approaches. Aim 2: We will determine how ML NK cells edit the phenotype of leukemia. Here, we will delineate how AML resistance to ML NK cells is distinct from AML resistant to cNK based upon differential KIR sensitivity and LAG-3 expression using genomic analysis, flow cytometry, and mechanistic LoF in vitro and murine xenograft models. Together, these findings lay the groundwork for Dr. Foltz’s research career on the mechanisms underlying NK cell clinical efficacy.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY I am an early career investigator in the fields of pharmacoepidemiology, population health, and addiction psychiatry. My overarching career goal is to become an independently funded researcher dedicated to investigating the real- world effectiveness of addiction medications so that we can address the gaps of the clinical trial literature with big data and reduce overdose. In this K08 award, I am interested in studying the comparative effectiveness and safety of FDA- approved pharmacotherapies (buprenorphine, methadone, naltrexone) to treat opioid use disorder during pregnancy, and assessing how the geographic landscape of treatment availability may be associated with their effectiveness against overdose across all American communities. The K08 award will support my early career training in the use of population health models, target trial emulation, and causal inference methods; address my mentorship and career development needs; and enable me to obtain the data required for my continued transition toward independence. In the proposed K08 research study, I will address the ongoing yet preventable epidemic of OUD-related overdose during pregnancy across states, geographies, and regions (urban vs rural). The specific aims of the study are (1) to evaluate access to opioid use disorder treatment for pregnant patients and identify communities, states, and regions with the highest rates of overdose and availability of FDA-approved pharmacotherapies; (2) to emulate a clinical trial using observational data to assess the comparative effectiveness of addiction treatments across communities in the U.S.; and (3) to examine population-level determinants of overdose as mechanisms underlying treatment outcomes during pregnancy. I will use a target trial emulation-based approach to achieve these aims, enriching existing Medicaid claims data with zip code-derived area-based characteristics to assess how population-based factors might influence treatment access and outcomes. The training and research experience I gain during the proposed K08 award period will be instrumental to my career trajectory, and the data generated in the proposed K08 study will be foundational to my planned applications for independent R01 funding, along with supporting NIDA's goal of developing new and improved strategies to prevent drug use and its consequences.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT The precise pathophysiology of Alzheimer’s disease (AD) remains unknown, but the development of amyloid- beta (Aβ) plaques is thought to be a key initiating event preceding the formation of neurofibrillary tangles and clinical disease. Anti-Aβ monoclonal antibodies (mAb) are the first disease modifying therapies approved for AD, but clinical trials showed significant and potentially fatal dose-limiting side effects, including amyloid-related imaging abnormalities (ARIA) that can manifest as edema and microhemorrhage. Peripheral monocytes/macrophages enter the brain and associate with Aβ plaques in the APP/PS1 transgenic mouse model of AD. These cells can reduce plaque load in vivo despite making up a small percentage of plaque-associated macrophages, suggesting that a better mechanistic understanding of the recruitment and function of these cells in the AD brain may improve anti-Aβ therapies. In preliminary work, a chimeric antigen receptor (CAR) was expressed in macrophages (CAR-Ms) containing an extracellular Aβ binding domain and an intracellular Fc receptor signaling domain to enhance the ability of these cells to phagocytose Aβ. Aβ CAR-Ms reduced plaque load ex vivo on amyloid-laden APP/PS1 brain slices, and in vivo when injected into the hippocampus of APP/PS1 mice; however, plaque reduction was confined to the region immediately surrounding the cell injection site and CAR-M migration was minimal, limiting its efficacy. The objective of this proposal is to enhance the efficacy of Aβ CAR-Ms by identifying factors that promote the infiltration of peripheral monocytes into the brain and their migration within the brain parenchyma. This proposal will test the hypothesis that improving brain infiltration and migration of Aβ CAR-Ms will improve Aβ clearance in aged APP/PS1 mice without increasing ARIA. An important theoretical advantage of CAR-M therapy compared to mAb therapy is the active degradation of plaque material, potentially mitigating concerns for ARIA. Aim 1 will determine whether CX3CR1 overexpression allows CAR-Ms to better infiltrate the brain from the periphery and characterize brain-penetrating CAR-Ms. This aim will further investigate the effect of Aβ CAR-Ms on the composition and phenotype of the brain cellular microenvironment. Aim 2 will compare the safety and efficacy of CAR-M therapy to mAb therapy in APP/PS1 mice. While mouse models do not replicate the entire spectrum of human ARIA, the frequency of microhemorrhages after CAR-M or mAb treatment will be quantified as a measure of ARIA-like phenomena. These studies will further develop and characterize a novel form of AD therapy and provide insights in basic macrophage biology. I will pursue these studies under the guidance of a team of physician-scientists, including a sponsor with expertise in CAR and macrophage biology, and a co-sponsor with over two decades of experience in AD mouse modeling. My training plan incorporates inter-departmental expertise and core facilities in the setting of Washington University’s prestigious MD and Immunology PhD programs, and I am confident that completion of this fellowship will provide me with the skills and career competencies necessary to develop into an independent physician-scientist.

NIH Research Projects · FY 2025 · 2024-08

Abstract Opioid use disorder (OUD) is rare (2%) but associated with extraordinarily high morbidity and mortality. Opioids are the primary contributors to the soaring numbers of U.S. drug overdose deaths, with increasing co-use of psychostimulants exacerbating the burden. Alongside this opioid crisis, are increasing rates of cannabis use, a drug that is now legal for recreational use in 24 states. Early cannabis use onset (eCan) has been documented in cross-national, cross-sectional, and longitudinal studies to increase the likelihood of using and misusing both cocaine/psychostimulants and opioids. Shared genetic vulnerability has been suggested as a mechanism undergirding both eCan and OUD, although it has not been estimated. However, even within discordant twin pairs, twins who endorse eCan more commonly report stimulant misuse and OUD, when compared to their co- twins (each at 4 times increased odds), suggesting that genetic factors alone do not explain this association. Also, rodents pretreated with delta-9 tetrahydrocannabinol during adolescence show variable behavioral and epigenetic responses to opioids and cocaine, suggesting that eCan may modify sensitization to these drugs. Yet, due to the rarity of OUD in samples that typically assess eCan and the absence of eCan measurement in large biobanks from which OUD data are drawn, a detailed examination of the association between eCan and opioid involvement is pending. In this R21 data analysis proposal in response to NOT-DA-22-003, using data from multiple deeply phenotyped samples, predominantly those ascertained for substance use disorders (N~138,000), we examine the association between eCan and opioid involvement. In aim 1, we estimate the association between early cannabis use (eCan) and proximal (e.g., initial subjective responses, indexing sensitization) and distal aspects of opioid involvement (e.g., OUD criterion count), and whether associations are modified by co-occurring stimulant (non-prescription, including cocaine) misuse. In aim 2, we test whether eCan and OUD (including overdose in the presence of psychostimulants) share an architecture of common genetic influences and parse evidence for non-genetic, putatively causal influences of eCan on opioid involvement using both genomic causality and data from pairs of twins and siblings discordant for eCan. In aim 3, we estimate the independent role of Adverse Childhood Experiences (ACEs) on eCan and opioid involvement and its interplay with genetic susceptibility in worsening the likelihood of OUD in those who start using cannabis at an earlier age. Understanding the relationship between eCan and specific aspects of opioid involvement provides insights into whether future changes in cannabis use might exacerbate OUD, and establishes targets for experimental and mechanistic studies. If eCan modifies opioid sensitization and increases likelihood of OUD, or is correlated with opioid overdose death, then heightened vigilance is urgently needed to delay onsets amidst efforts to legalize the drug.

NIH Research Projects · FY 2024 · 2024-08

Project Summary Current paradigms of drug development have made remarkable progress in treating and preventing disease by effecting the function of a single target enzyme, or receptor with specificity. However, many chronic diseases like type 2 diabetes, Alzheimer’s disease, and cardiovascular disease are multifaceted and may require alterations in multiple pathways, or delivery to intracellular targets, to effectively treat the condition. For this reason, extracellular vesicles (EVs) have gained attention as potential therapeutic carriers and endogenous modulators of disease. EVs are nano-sized vesicles produced in all cells and carry macromolecules that are capable of modulating multiple pathways simultaneously in the receiving cell. Their cargo includes miRNAs, mRNA, DNA, signaling proteins, enzymes metabolites and receptors. Mesenchymal stem cells (MSCs) have been extensively used as the parent cells for therapeutic EVs as the cargo of these cells is broadly protective. Many groups have engineered MSC EVs to carry therapeutic compounds through permeabilization techniques which rely on passive diffusion of compounds into isolated EVs. However, the current engineering techniques are exceptionally inefficient, which is a major factor in preventing the translation of engineered EVs into the clinic. In this proposal we utilize vascular endothelial cells (ECs) to actively package desired cargo into EVs. This is possible through a novel transcytosis-like process we recently published. We found that ECs take up exogenous albumin-bound material from the cell culture media, load the endocytosed material into newly formed EVs, and export those EVs at the basolateral side of the cell. Interestingly, the released EVs seem to target specific cells in the tissue. Therefore, we have designed experiments to test and optimize the capacity of ECs to load EVs with exogenous albumin-bound miRNA, peptides, and drugs. We will test the therapeutic efficacy of customized EVs in an in vitro and in vivo myocardial infarction mouse model system. We expect that utilizing ECs will allow for extremely efficient, active loading of EVs, unmatched targeting to the desired cell type and improved cargo offloading efficiency in receiving cells. This work has the potential to transform the EV engineering field and bring us closer to clinically applicable EV- based therapeutics.

NIH Research Projects · FY 2025 · 2024-08

PROJECT ABSTRACT This proposed career development award will provide Dr. Danielle Alfano, MD with targeted mentored training to ensure she develops into an independent researcher utilizing both experimental approaches, mouse models, and “omics”, to probe how specific pathogens harness ADAM10 leading to the dysregulated endothelial and host response in sepsis. Sepsis is a complex syndrome defined as a ‘dysregulated host response to infection leading to life-threatening organ dysfunction’. While many studies in the field have focused on an aberrant host immune response as the instigator of severe sepsis, all efforts have failed to translate this into new therapies. The endothelium plays a critical role in the host response to infection and organ injury. We have recently demonstrated that ADAM10, the eukaryotic receptor for S. aureus α-toxin, acts a molecular specifier of sepsis, mediating mortality and endothelial injury to a diverse subset of pathogens. However, the precise molecular mechanisms remain poorly understood. The proposed research plan aims to close key knowledge gaps regarding how specific pathogens harness ADAM10 in disease and the molecular mechanisms of endothelial injury. To do so, the PI will leverage multiple live pathogens and unique mouse lines to fully characterize the nature of ADAM10 functions that contribute to sepsis progression. The PI aims to 1) understand the molecular mechanisms by which diverse pathogens activate endothelial ADAM10, 2) characterize ADAM10 specific substrates released during systemic infection, and 3) examine how an ADAM10 SNP polymorphism may confer increased risk of lethal disease. The proposed 5-year career development and training plan incorporates strategically designed didactic learning, mentored practical training, and career advising to complement the PI’s expertise in ways that are critical to completion of her research and career goals. The specific career development goals outlined in this application include developing mechanistic expertise in 1) vascular biology and intracellular signaling pathways; 2) host-pathogen interactions; and 3) proteomics. She will be training at WUSM, a world- class center for basic and translational research and an excellent environment for physician- scientist training with experts in all aspects of the proposed training. She will be closely mentored by Dr. Juliane Bubeck Wardenburg, an expert in S. aureus, ADAM10, host-pathogen interactions, and immunology. The long- term goal is to provide Dr. Alfano with the skills required to become an independent, R01- funded faculty member working to study pathogen-specific molecular determinants of sepsis and factors that confer increased risk of life-threatening sepsis to certain populations to provide novel insights into future therapeutic developments.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract This proposal encompasses a 5-year research and training plan to support the transition of Dr. Wattenberg to an independent research career. Dr. Wattenberg’s long-term goals are to lead an independent R01-funded research program at an academic institution, conducting research focused on defining relevant cancer immunobiology. To achieve this, Dr. Wattenberg’s short-term goals are to gain expertise in the areas of mouse modeling, transcriptomics and clinical trials. These efforts will be supported by the K08 award and through mentorship provided by Dr. Gregory Beatty and a mentorship committee consisting of successful senior scientists. Additional formal training will be provided through a didactic curriculum, including workshops and courses. Research will be conducted at the University of Pennsylvania, which provides comprehensive institutional resources and a robust research environment for the training of physician-scientists. The proposal focuses on defining how immune memory in cancer is initiated and disrupted. Studies will be performed using clinically relevant mouse models of cancer and patient samples. Immune memory is crucial for durable tumor control induced by immunotherapy. However, durable tumor responses are rare in patients. Better understanding of how immune memory develops is needed to inform novel immunotherapy strategies. Preliminary data show that activation of conventional dendritic cell (cDCs) subtypes triggers immunological memory in mouse models of pancreatic cancer. Further, immunological memory in this model is dependent on CD4+ T cells and not CD8+ T cells - the prototypical effector cells of the immune system. Additional prior work shows that systemically elevated inflammatory proteins associate with poor clinical outcomes to cDC targeted immunotherapy (CD40 agonist) in patients with pancreatic cancer. Moreover, complementary studies in mouse models show the inflammatory cytokine IL-6 to associate with cDC dysregulation. These findings suggest that inflammatory proteins drive cDC dysfunction, limiting productive immunosurveillance. It is the central hypothesis of this proposal that distinct cDC subtypes coordinate CD4+ T cell memory, which is necessary for durable tumor immunosurveillance, and that cancer-associated inflammatory proteins drive T cell immune evasion by dysregulating cDC subtypes. Dr. Wattenberg will investigate this hypothesis in the following 3 aims. Aim 1. Determine the impact of cDC subtypes on tumor specific CD4+ T cell memory. Aim 2. Determine the role of CD4+ T cells in coordinating anti-tumor immunological memory. Aim 3. Define the mechanisms by which cancer- associated inflammatory proteins drive cDC fate. Successful completion of this proposal will provide fundamental insights into cancer immunobiology and identify novel immunotherapy strategies to improve outcomes for patients with cancer. Further, the mentorship and training provided by this proposal will support Dr. Wattenberg’s transition to independence leading a translational research group.

NIH Research Projects · FY 2025 · 2024-07

Mechanism of neutrophil dysfunction by Plasmodium falciparum secreted histidine-rich protein II People with malaria infection caused by Plasmodium falciparum are significantly more susceptible to invasive bacterial infections during and in the weeks following acute malaria infection. Plasma from infected patients can modulate neutrophil function, a critical aspect of the immune response for controlling bacterial infection. Interestingly, neutrophils isolated from children with malaria have an impaired oxidative burst even at 4 weeks following malaria infection. Proposed models of neutrophil dysfunction suggest a role for heme induction of heme oxygenase 1, an enzyme known to reduce the production of reactive oxygen species in neutrophils by down- regulating expression of NADPH oxidase subunits. However, heme does not remain elevated after malaria infection and does not explain the persistent neutrophil dysfunction. We propose a model that incorporates histidine-rich protein II (HRPII), an abundant secreted parasite protein that circulates as a nanoparticle. Notably, HRPII has a long half-life and persists in children with malaria for up to 4 weeks. We previously showed that heme-laden HRPII (HRPII:heme) delivers a high load of intracellular heme, inducing reactive oxygen species (ROS), inflammation, and cellular junction disruption in vascular endothelial cells and causes vascular leakage in mice. In preliminary studies using a model human neutrophil-like cell line (dHL60), we have found that HRPII:heme, unlike free heme, does not directly stimulate the oxidative burst. Pre-treatment with HRPII:heme prior to dHL60 stimulation with PMA results in decreased oxidative burst within a few minutes. It also causes a slow but dramatic induction of heme oxygenase 1. The goal of this proposal is to identify the molecular mechanisms by which neutrophils sense HRPII:heme and the signaling pathways that are required to impair oxidative burst activity. Our first aim utilizes targeted molecular blockade in dHL60 cells of known pathways of HRPII cellular effects and neutrophil pathways involved in oxidative burst signaling. Our second aim will utilize an unbiased approach with transcriptome profiling (RNA-seq) of dHL60 treated with HRPII and a genome-wide CRISPR/Cas9 knockout screen to determine genes essential for HRPII effects. The proposed work will provide insight into how HRPII affects neutrophils and, in time, potential therapeutics to prevent invasive bacterial infections in patients with malaria.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Bone fractures occur in more than 25 million Americans per year, and 5-10% of those fractures fail to heal. Fractures that show delayed or failed progression to healing account for the majority of patient disability and costs. Thus, there is an urgent need to better understand the biology of bone healing, and to use that knowledge to develop new diagnostics and therapeutics. We are seeking support for the 18th Biennial conference of the International Section of Fracture Repair (ISFR), a two-day meeting in Montreal. In 2017, the ISFR was incorporated as a section within the Orthopaedic Research Society (ORS), thereby leveraging the outreach of the largest scientific organization in the world dedicated to orthopaedic research. The ISFR is dedicated to the advancement and exchange of the most current scientific ideas and research findings on fracture repair and its application to the improvement of patient care. Our mission is be the premier forum to integrate and present cutting-edge ideas related to clinical, translational, and basic-science research of fracture healing. With an organizational theme of “The Future of Fracture Repair”, the main sessions of the meeting will present forward-looking research and issues, in addition to challenges facing the trauma surgeon now: Day 1) cross-talk of bone with other tissues, the future of post-fracture rehabilitation, and next-generation outcomes assessment in fracture healing; Day 2) nonunion, geriatric fractures and polytrauma. In a new partnership intended to promote clinical participation and clinician-scientist interaction, the ISFR Meeting will be held in conjunction with the 2024 Annual Meeting of the Orthopaedic Trauma Association (OTA). The first day of the ISFR conference has been planned to provide basic and translational content of interest to researchers, clinicians and industry professionals. The second day has been jointly organized by the ISFR and the OTA, with a focus on the latest research on clinically relevant topics. In total, we will have an invited keynote speaker, 27 invited session speakers, and slots for 24 abstract-selected podium presentations. We anticipate an additional 30-40 abstracts selected as posters. The Program Committee is highly focused on fostering an inclusive environment, and has strived for a diverse roster of invited speakers, in terms of gender, career stage and URiM representation. Surgeons, biologists, engineers, and policy makers will attend the meeting and be drawn from academia, health-care, government, and industry. A variety of activities will be focused on career development and networking for trainees in the bone healing field.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY/ABSTRACT – In patients with gastric cancers who are clinically classified as HER2- positive, antibody-drug conjugates (ADC) prolong progression-free and overall survival. However, not all gastric tumors benefit from these therapies, or even those who initially respond inevitably develop resistance over time. Complementary biomarkers and methods are therefore needed to identify patients that will respond or develop resistance to HER2-targeting ADC therapies. Preclinical data we obtained in HER2 heterogeneous gastric patient-derived xenografts (PDXs) demonstrate that tumoral sphingosine-1-phosphate receptor (S1PR1) associate with tumors’ response to ADCs. Here, we will validate a S1PR1-targeted imaging approach (that is already used in the clinics for non-cancer applications) that allows quantitative measurements of S1PR1 to evaluate the early response to ADC treatment in mouse models of gastric cancer. We will correlate HER2 membrane availability, tumoral S1PR1, and the presence of genetic alterations with gastric tumor response to ADCs. We will perform randomized imaging and therapeutic studies in gastric PDXs to identify biological features that confer tumor sensitivity and resistance to ADCs. Aim 1 will use molecular imaging approaches involving positron emission tomography (PET) to further investigate the role of S1PR1 in ADC efficacy. Our innovative approach can quantitatively measure ADC-tumor binding through immunoPET and S1PR1 through a radiolabeled small molecule in the same subject and in real-time in gastric PDXs. Our studies will show that our imaging approaches, combined with analyses at the tissue level, can quantitatively detect different levels of early response to ADC therapy in gastric tumors, providing a strong impact. Aim 2 will further establish PET to determine the potential of modulating tumoral S1PR1 to reverse ADC resistance in gastric tumors. We will measure changes in HER2 and S1PR1 in response to treatments that enhance ADC efficacy. For each study, we will correlate the magnitudes of changes in our imaging biomarkers to a longer- term therapeutic outcome, as well as determine which combinations of multiple biomarkers improve the evaluation of early treatment response. This project could provide an excellent foundation for many future investigations, including the clinical translation of PET imaging approaches to predict and monitor tumor response to ADCs and the potential broader application to other membrane receptors and heterogeneous tumors. The long-term translational objectives of the studies proposed are to establish a foundation for a clinical study using imaging approaches of antibody-tumor accumulation and S1PR1 as predictive biomarkers of tumors response to HER2-targeting antibody drugs.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Alzheimer’s disease (AD) is a devastating and complex neurodegenerative disorder with a crushing social and economic burden currently affecting more than 6 million patients in the US. With the number of affected individu- als projected to increase, identifying disease-modifying treatments and interventions for AD remains one of the most critical undertakings in modern biomedical research. A critical step to the development of these interven- tions involves characterizing the genes and pathways mediating AD risk and resilience. The objective of this proposal is to explore the modulation of AD genetic risk by age and examine how caloric restriction (CR), a lifespan-increasing intervention, can be harnessed to counter AD risk. The proposal will utilize state-of- the-art single-nuclei multi-omic functional genomic analyses of healthy and AD human brain tissue, as well as transgenic AD mouse models, to perform deep molecular characterization of AD risk and resilience across the genetic, epigenomic, and transcriptomic layers. Aim 1 will dissect non-coding AD genetic risk through intensive multi-omic molecular profiling of healthy and AD brains, allowing the nomination of causal variants, cell types, and pathways mediating risk. Aim 2 will identify protective mechanisms in cognitively healthy, long-lived individ- uals, including centenarians, and determine whether AD resilience manifests by maintaining a “young” state or by compensating for detrimental age-related effects and AD genetic risk. This will involve comparison across a broad age spectrum, including molecular profiles of young controls (aged 18-35) and AD patients. Aim 3 will characterize the interplay between CR and neurodegeneration in genetically diverse AD mouse models. This will provide insight into the pathways mediating lifespan-increasing interventions in the brain, their interaction with high-risk and resilient genetic backgrounds, and their overlap with AD risk and resilience pathways in hu- mans. Importantly, this proposal will directly address the connections between lifespan and AD resilience to provide a more nuanced understanding of the potential of CR and its mimetics as therapeutic strategies for AD. This proposal employs an innovative research approach combining observational and interventional study de- signs with state-of-the-art computational techniques. This approach facilitates concurrent human and mouse data analysis, expediting the discovery of molecular pathways mediating AD risk and resilience, with potential implications for developing novel AD prevention strategies. Under the mentorship of Dr. Harari and co-mentors Dr. Goate, Kaczorowski, Karch, and Lee, I will follow a rigorous training program to meet the aims of this K99/R00 award and transition into independent research leadership in the domain of AD and aging. This will include a focus on neurobiology, the biology of aging, mouse models of AD, bioinformatics, and professional development, achieved through courses, workshops, conferences, and advisory committee feedback. In summary, this pro- posal addresses a critical gap in our understanding of AD and aging, with the additional skills acquired during this award laying a solid foundation for my future independence in the molecular biology of AD.

NIH Research Projects · FY 2025 · 2024-07

Project Summary This proposal is a five-year career development plan that will support my transition to an independent career as a neuroscientist and neuro-ophthalmologist focused on understanding the mechanisms of neurodegeneration in the visual system and developing therapies for vision restoration. I am currently an Instructor in the Department of Neurology. Under the mentorship of Dr. David Gutmann, MD, PhD, and with the guidance of my scientific advisory committee, I will receive didactic and practical training to expand my scientific and clinical expertise, as well as improve my skills in collaboration, communication, lab management, grantsmanship, and leadership needed for an independent scientific career. In addition, I will build upon my preliminary experiments to delineate the mechanisms underlying estrogen-mediated (extrinsic) and cyclic-AMP (cAMP)-mediated (intrinsic) vulnerability of retinal ganglion cells (RGCs) that collectively culminate in RGC death and vision loss in an authenticated mouse model of optic pathway glioma (OPG). Neurofibromatosis type 1 (NF1) is a cancer predisposition syndrome in which 15-20% of children develop an OPG, a low-grade astrocytoma of the visual pathway. Of these affected children, 30-50% will experience vision loss due to RGC death. Moreover, girls with NF1-OPG are 3-5 times more likely to require treatment due to vision loss than boys, despite a relatively equal incidence of OPGs in both sexes. These findings suggest that vision loss from NF1-OPG is controlled by mechanisms that are sexually dimorphic. Leveraging authenticated mouse models of Nf1-OPG developed by the Gutmann laboratory, I identified estrogen-activated glial production of interleukin 1β (IL-1β) as a possible extrinsic mechanism for RGC death due to Nf1-OPG. Building upon previous findings demonstrating that defective cAMP production predisposes Nf1-mutant RGCs to injury, I also identified cAMP-dependent protein kinase inhibitor α (PKIα) as a potential mediator of the intrinsic RGC vulnerability in the setting of Nf1-OPG. Based on these exciting preliminary results, I hypothesize that estrogen- mediated glial expression of IL-1β is neurotoxic to Nf1-mutant RGCs with a pre-existing vulnerability to death conferred by reduced cAMP-dependent survival signaling. In this proposal, I have designed experiments to (1) define the necessity of estrogen-induced IL-1β in Nf1-OPG-mediated RGC death, (2) determine whether PKA inhibition heightens RGC vulnerability in the setting of Nf1-OPG, and (3) execute preclinical studies to evaluate estrogen suppression and cAMP restoration as potential neuroprotective strategies. The goals of the proposed experiments aim to (1) expand our understanding of molecular mechanisms underlying RGC death and vision loss in Nf1-OPG, (2) identify targets for the development of vision restorative therapies for children with vision loss due to NF1-OPG, and (3) lay the groundwork for my future studies and career development as a neuro- ophthalmologist/neuroscientist focused on extrinsic and intrinsic mechanisms of neuronal injury in diseases of the visual system.

NSF Awards · FY 2024 · 2024-07

With the support of the Macromolecular, Supramolecular and Nanochemistry Program and the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Bryce Sadtler of Washington University in St. Louis will use advanced microscopy to understand how the structures of single-atom photocatalysts change over time when they catalyze chemical reactions to produce fuel. Photocatalysts can absorb sunlight to initiate valuable reactions for energy production and environmental remediation, such as the reduction of carbon dioxide into a fuel source like methanol. Single-atom photocatalysts are a relatively new type of photocatalyst consisting of individually dispersed metal atoms on a semiconductor support. Single-atom photocatalysts have potential advantages over other types of photocatalysts, including increased activity compared with nanoparticles for fuel-forming reactions. However, it is currently very challenging to monitor how the structures of single-atom photocatalysts may change over time, and how those changes impact their efficiency. Professor Sadtler and his students will use single-molecule fluorescence microscopy to image individual catalytic reactions on the surface of single-atom photocatalysts; this technique will enable them to visualize whether the distribution of metal atoms changes over time. By identifying whether individual metal atoms or small clusters of atoms are more catalytically active, Sadtler and his team aim to design more efficient and stable single-atom photocatalysts to produce chemical fuels in a carbon-neutral manner. To broaden participation in these research activities, Professor Sadtler will host high-school teachers in his laboratory during the summer to conduct research on catalyst synthesis and characterization. Current synthetic methods used to make single-atom catalysts typically result in significant variations in the distribution of metal atoms and their coordination to the surface of the support; this structural heterogeneity produces different types of active sites and makes it difficult to elucidate structure/activity relationships based on ensemble measurements of catalytic activity. This project will use single-molecule fluorescence microscopy with chemically activated fluorogenic probes to quantify differences in photocatalytic activity at the nanoscale for dispersed metal atoms that are supported on semiconductor metal oxides. These studies can provide new insights into how heterogeneity of the nanoscale distribution of supported metal atoms controls the regions of the particle that are photocatalytically active. The utility of these experiments will be assessed by applying observations made at the single-molecule and single-particle levels to rationalize and predict structure/activity relationships measured at the ensemble level for the reduction of carbon dioxide using single-atom photocatalysts. 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 2026 · 2024-07

The ability of bacteria to adhere to each other and both biotic and abiotic surfaces is key to their ability to cause infection and persist in the environment. Bacteria can adhere using proteinaceous adhesins, including fibrillar adhesins. Key characteristics of fibrillar adhesins include: (i) they are extracellular, surface-associated proteins, (ii) they possess an adhesive domain as well as a repetitive stalk domain, and (iii) they are either a monomer or homotrimer (i.e., identical, coiled-coil) of a high molecular weight protein. Fibrillar adhesins are widely abundant in bacteria with at least 26% of all UniProt bacterial reference proteomes containing predicted fibrillar adhesin-like proteins. They provide a range of functionalities to the bacterial surface including adherence to host tissues, and in many instances, fibrillar adhesins are key to structuring biofilms, which are aggregated bacterial communities that cause chronic, difficult-to-treat infections. Thus, strategies to block fibrillar adhesin-mediated bacterial adhesion and biofilm formation are desirable therapeutic targets. However, despite the wide-abundance and prominent role in infection that fibrillar adhesins play, we have limited understanding of fibrillar adhesins because their massive size and repetitive sequences have prevented structural and molecular biophysical studies of full-length, intact proteins. Instead, structures and biomolecular interactions of well-behaving domains of some fibrillar adhesins have been determined, and in a handful of cases, the domain structures have been stitched together with a heavy reliance on homology modeling. The lack of structural insight into fibrillar adhesins is problematic because protein structure is directly related to function. As it currently stands, the field lacks key understanding of a widely used mechanism of bacterial attachment, and we are missing out on opportunities to rationally design therapeutics to prevent bacterial attachment to abiotic and biotic surfaces and biofilm formation. To address this knowledge gap, we will develop novel approaches to elucidate the structure and interactions of a model fibrillar adhesin, the Pseudomonas aeruginosa biofilm matrix protein called CdrA, and then explore the impact of these features on fibrillar adhesin function. The MIRA award will enable the PI (Reichhardt) to dedicate greater time and resources to addressing this knowledge gap. Looking to the future, this proposed research will open the path to the structural and molecular biophysical studies of other fibrillar adhesins as well as other high molecular weight or repetitive proteins (e.g., eukaryotic extracellular matrix proteins).