Northwestern University

NIH Research Projects · FY 2025 · 2025-09

Acute myeloid leukemia (AML) is a hematologic cancer with a generally poor prognosis, and mutations in TP53 define one of the most adverse risk molecular subsets. National and co-operative group datasets suggest that sociodemographic characteristics (social deprivation index, education, poverty, insurance) influence survival in AML. We established the multi-institution Chicago AML Registry that has collected demographic and clinical data on 822 AML patients. In our published analysis, composite census tract-based socioeconomic measures of disadvantage and affluence were found to be potent mediators of observed AML survival differences. These tract-based measures may be markers for specific aspects of patients’ social and physical environments (SPE) that contribute etiologically to poor AML outcomes. Our preliminary data confirms an increased prevalence of adverse genomic characteristics in AML patients living in adverse SPE, specifically TP53 mutations and complex cytogenetic abnormalities. In Aim 1 we will develop a neighborhood stress scale, incorporating area deprivation index and gun violence data, and link this to adverse genomic characteristics of AML as well as more distal outcomes including treatment response and relapse-free and overall survival. We also develop a prospective study of preleukemic patients bearing small clones of TP53 mutated hematopoietic stem cells and examine the effects of self-reported social stress on inflammation and clonal evolution. We next address mechanisms by which factors within the SPE drive TP53 mutant clonal expansion. Our preliminary data confirm that an increased burden of social stressors results in activation of the innate immune system. Leveraging models of social stress in transgenic murine models of mutant TP53 Clonal Hematopoiesis, as well as serial patient samples we will study dynamic interactions between social environment, ancestry, and clonal trajectories in AML. Importantly, our analyses will consider both self-identified race/ethnicity (SIRE) and genetic ancestry based on ancestry informative markers (AIMs) to appreciate the overlapping, but independent effects of social constructs and genetic ancestry that may influence inflammation and immune response. Our preliminary studies suggest that the NLRP1 inflammasome/IL-1β axis may represent a mechanistic link between social stressors and TP53 mutant clonal evolution and we will develop xenografts from AML patients from a range of social backgrounds to determine the therapeutic impact of targeting the NLRP1 inflammasome/IL-1β axis. The goal of this project is to triangulate registry data, patient samples and animal models to establish social stress as a biologic determinant of clonal trajectories in AML development.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Injury is the most common cause of death in people 46 years of age and younger. Bleeding from injury (trauma-induced hemorrhage) is the second leading cause of injury-associated death and the most common cause of preventable death. Annually, 70-100,000 Americans suffer a preventable death from trauma-induced hemorrhage. These deaths could be prevented were patients to receive timely care (e.g., hemostatic resuscitation and hemorrhage control procedures within two hours of injury) in hospitals that specialize in injury care (e.g., high level trauma centers). These high-level trauma centers have the equipment, blood products, medications, massive transfusion protocols and staff 24 hours a day, 7 days a week to provide hemostatic resuscitation and hemorrhage control procedures. These resources and services are not available at non- specialized hospitals. Management of bleeding requires timely procedures to stop the bleeding as well as medications and blood transfusions to prevent blood from thinning (trauma-induced coagulopathy). Our previous work demonstrated that the most common reason patients were not quickly transferred (retriaged) from non-specialized hospitals to another specialized high-level trauma center was because clinicians did not know who should be retriaged. There are no national retriage guidelines. Our literature review demonstrated only 22 out of 50 states in the United States have any retriage guidelines. Even among states that have retriage guidelines, most guidelines are very vague (e.g., hypotension requiring blood transfusions). This application’s broad, long-term objective is to improve the timeliness of care of trauma-induced hemorrhage. We propose to comperehensively determine who should be quickly retriaged to specialized high-level trauma centers, and determine if state-of-the-art approaches to informing retriage decisions could improve timliness of treatment and reduce death rates. This study’s first specific aim will identify which patients and injuries have the greatest reduction in death rates upon retriage from non-specialized to specialized high-level trauma centers using Causal Graphical Models. The second aim will understand which patient injury and context attributes multi-speciality injury experts prioritize when making retriage choices using Discrete Choice Experiments. The third aim will determine if a protocol providing retriage decision support [PRotocol for OpMTimal reTRiage [PROMTR)] could improve time to treatment and death rates compared to usual care using Discrete Event Simulation. Upon demonstrating efficacy in improved timeliness of treatment and death rates in this simulation study, our future direction will be implementing and evaluating effectiveness of PROMTR in a Hybrid Type II Cluster Randomized Controlled Trial. This line of research has high potential to improve and standardize existing retriage practices, resulting in the reduction of preventable injury-associated death from bleeding.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT As the global population ages, the prevalence of Alzheimer’s disease and related dementias (ADRD) is on the rise. This poses significant challenges to healthcare systems worldwide. Primary Progressive Aphasia (PPA) is a clinical ADRD dementia syndrome characterized by progressive decline in comprehension and expressive language skills. While the primary approach for addressing language and communication impairments in PPA involves non-pharmacological intervention administered by a speech-language pathologist, access to care can be difficult due to a limited number of clinicians and lack of evidence-based interventions. While technology- supported interventions have the potential to improve access to care, promote social participation, ameliorate communication skills, and improve emotional well-being, there has been no systematic exploration of how individuals with PPA and related dementias use web applications. This study will address this gap by conducting a comprehensive analysis of an existing web application used in the Communication Bridge-2 randomized controlled trial (RCT), to provide insights into the usage, feasibility, and usability of such technologies in supporting communication for individuals with PPA and related dementias. Aim 1 will establish practical use guidelines for the existing web application. We will analyze retrospective app user data and post- study interview transcripts from Communication Bridge-2, an NIH stage 2 RCT of speech-language therapy for PPA. We will categorize user groups (frequent vs. nonfrequent users) and determine which clinical and demographic factors (e.g., sex, age, aphasia severity) correlate with greater app engagement. This will allow us to determine which groups are using the app vs. which are not and may require additional engagement strategies. For Aim 2, we will identify evidence-based steps to optimize the existing app for a broader audience of PPA and related dementias. Through semi-structured interviews with individuals with PPA and their communication partners, we will identify implementation barriers to technology-supported intervention and propose necessary changes to improve user engagement. This will allow us to evaluate what app features users like, to identify app limitations, and to propose modifications to the app to improve user experience in this population. This project will occur within the Healthy Aging & Alzheimer's Research Care Center at the University of Chicago, where I will work with data from the only large-scale RCT for language and communication in PPA, as well as individuals with PPA referred from around the world. Findings have the potential to optimize technology use to maximize communication skills, quality of life, and overall care for those within the ADRD spectrum.

NIH Research Projects · FY 2026 · 2025-09

There are 24,000 stillbirths annually in the United States. In population-level studies, unexplained stillbirths are 30-40%. However, in medical-center based studies with more detailed work-up, the rate is <10%. These studies consistently show that placental histopathology is the most informative datum. This may seem counterintuitive – unlike whole exome sequencing or hemoglobin F flow cytometry, histopathology is widely available. The limitation is the sparsity of human experts. There are around 100 expert perinatal pathologists in the United States, almost all in urban academic medical centers. The situation is unlikely to improve – most projections show the total number of pathologists going down. Our solution is to develop machine learning (ML) models that can make the key placental histopathologic diagnoses in stillbirth. Placental findings may reinforce the significance of information already known, such as evidence of maternal vascular injury in patients with hypertensive disorders of pregnancy. Alternatively, placental findings may be critical, but have no established way of diagnosing them before delivery, such as histiocytic intervillositis. We will test these models in a group of >1500 pregnancies that ended in stillbirth. We will perform detailed abstraction of clinical, obstetric, and laboratory information. Pathologists and maternal-fetal medicine experts will confer on classifying the cause of demise. We will test the importance of placental diagnoses in identifying the anomaly leading to demise and evaluate whether machine learning diagnoses give meaningful information on the causes of demise. To improve generalizability, we will test our model using a set of 500 stillbirths from the Mayo Clinic. Better identifying the cause of demise has immediate and long-term benefits. Understanding what happened is an important part of grieving for patients and families experiencing stillbirth. The benefits are magnified if patients attempt a subsequent pregnancy, since their risk can be better delineated. Knowing the cause of a prior stillbirth can allow personalized treatment. Researchers focusing on specific causes of stillbirth need to identify patients at risk of that cause and to target them for treatment. In the US, stillbirths are reviewed in multidisciplinary conferences. The models developed in this study can improve the work of pathologists in these conferences and, potentially, support decision making in these groups. With sufficient improvement, these models can be piloted in lower-resource settings as full replacement for pathologists or conferences.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Mitochondrial dysfunction is a well-established cause of neurologic and psychiatric disease. The brain is the body’s most energetically demanding tissue and is especially vulnerable to metabolic challenges such as hypoglycemia, ischemia, and mitochondrial disease. One such mitochondrial disease is caused by defects in the mitochondrial enzyme MDH2, which catalyzes the final step in the TCA. Patients with mutations in MDH2 develop infantile encephalopathy and epilepsy. The association between mitochondrial dysfunction and neurologic disease is often attributed to a deficiency in ATP. However, in addition to ATP, the mitochondrial TCA generates biosynthetic intermediates like α-ketoglutarate (α-KG) which is used to synthesize the neurotransmitter gamma- aminobutyric acid (GABA). GABA is the brain’s primary inhibitory neurotransmitter, and decreased GABA signaling underlies neurologic and psychiatric disorders such as major depressive disorder, anxiety disorders, schizophrenia, autism spectrum disorder, and epilepsy. The research proposal aims to elucidate the critical role of mitochondrial MDH2 in ATP and GABA production within GABAergic neurons. The hypothesis predicts that MDH2-deficient GABAergic neurons can produce adequate ATP through glycolysis but fail to synthesize GABA, leading to impaired GABA signaling without neuron death. The proposal tests the hypothesis with two specific aims. The first aim investigates whether MDH2 is essential for ATP production in GABAergic neurons. This involves studying the bioenergetic consequences of MDH2 loss in human iPSC-derived GABAergic neuron cultures and a mouse model with MDH2 selectively knocked out in GABAergic neurons. The second aim explores the necessity of MDH2 for GABA synthesis using techniques such as metabolomics, RNA sequencing, isotope tracing, and spatial metabolomics. Additionally, the proposal examines the potential of α-ketoglutarate (α-KG) as a supplementary substrate for GABA synthesis to mitigate MDH2 deficiency. This research seeks to uncover the metabolic requirements for GABAergic signaling, providing insights into the connection between mitochondrial dysfunction and GABA-related diseases, and paving the way for new therapeutic strategies targeting GABAergic neuron metabolism.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT We will test the effectiveness of a technology-enabled strategy to optimize blood pressure among reproductive-aged women with hypertension receiving care in Federally Qualified Health Centers. Nearly 1 in 6 women of reproductive age have chronic hypertension (HTN) and are at increased risk of premature cardiovascular disease (CVD) and death. Women with HTN who become pregnant also face higher risks of maternal morbidity and mortality. Ensuring appropriate medication use can help young women achieve optimal blood pressure (BP) and reduce cardiovascular and reproductive risks. This includes not only selecting and adhering to appropriate antihypertensives (antiHTN) but also avoiding use of combined hormonal contraceptives which can increase BP. Yet few systems exist to improve medication use and BP in this high-risk population. In response, our team has worked with our FQHC partners to co-develop a multicomponent, technology-based strategy to promote the REproductive And Cardiovascular Health Of Underserved Patients with HyperTension (REACH-OUT). Our strategy seeks to promote safe antiHTN and contraceptive use among women with HTN and to create an infrastructure to monitor antiHTN adherence and BP to inform clinical decision-making. For clinicians, REACH-OUT includes [1] EHR-based clinical decision support that: a) elicits pregnancy intention in a non-judgmental manner, b) facilitates review of prescribed antiHTNs and contraceptives to identify contraindications, and c) prompts targeted counseling on medication safety, adherence, and BP for young women with HTN. For patients, REACH-OUT provides [2] educational materials, printed with after visit paperwork, to reinforce clinician counseling, [3] a BP monitor, training, and access to a patient portal-based tool where home BP measures can be recorded, and [4] a brief, portal-based survey to assess antiHTN use and ‘phenotype’ causes of poor adherence for clinic review. For patients who have difficulty monitoring their BP and adherence at home, [5] a patient navigator will provide tailored support and help troubleshoot any challenges. We will test REACH-OUT vs. usual care in a clinic-randomized trial among 12 FQHCs in Chicago, IL. We will enroll 350 English or Spanish-speaking, non-pregnant women on antiHTN therapy with elevated BP. Our aims are to: 1) Test the effectiveness of REACH-OUT, compared to usual care, to lower systolic BP, improve antiHTN adherence, and reduce use of contraindicated medications within 3 months; 2) Assess the reach, adoption, implementation, maintenance, and costs of REACH-OUT components; and 3) Explore the dose-response effect of REACH-OUT on BP, antiHTN adherence, and use of contraindicated medications over 12 months.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Human papillomaviruses (HPV) infect stratified squamous epithelia and link their productive life cycles to the differentiation of the host cell. HPVs infect cells in the basal layer and establish genomes as low copy nuclear episomes. When HPV infected cells migrate from the basal layer, they become arrested in G1, and a subset are induced to re-enter G2/M phases in the most differentiated layers to allow for productive viral DNA replication in a process referred to as amplification. Our recent studies have shown that activation of both the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) DNA damage repair (DDR) pathways is critical for viral amplification in differentiated cells. Furthermore, activation of ATR but not ATM, is required for stable maintenance of viral episomes in undifferentiated cells. Our studies further showed that HPV proteins activate these repair pathways by inducing high levels of DNA breaks in both cellular and viral DNAs that are caused by aberrant R-loops along with the action of topoisomerases. R-loops are trimeric nucleic acid structures that consist of a hybrid between RNA and its complementary DNA strand along with the displaced single strand DNA. These are stable structures that form at promoter as well as transcription termination regions. R-loops play important roles in the normal regulation of transcription initiation and termination however failure to resolve aberrant R-loops leads to DNA break formation. Our studies indicate R-loop levels are increased by 5 to 10-fold in HPV positive cells and maintenance of these high levels is necessary for viral replication and transcription as either decreasing or increasing the amounts leads to impaired viral functions. Furthermore, our work indicates that these enhanced R-loop levels also regulate expression of cellular pathways that are important for viral pathogenesis. E6 is responsible for the high levels of R-loops in HPV positive cells through its inhibition of p53. This application will investigate the mechanisms by which R-loop levels are increased in HPV positive cells and why this is critical for the viral transcription as well as replication. Specifically, we will examine the role R-loops play in differentiation-dependent amplification and late viral gene expression. In addition, the role of E6 in regulating R-loop levels through stabilization will be examined along with investigating which downstream targets of p53 effects are responsible for regulating R-loop levels. Finally, a role for the cytosine deaminase, APOBCEC 3B, in regulating R-loop formation in HPV positive cells will be addressed. Overall, the proposed studies will provide important insights into why R-loops play a critical role in the HPV life cycle.

NSF Awards · FY 2025 · 2025-08

The problem of understanding wave propagation is ubiquitous in mathematics and its physical applications: sound waves, light waves, ripples in the structure of spacetime, and the "wave-functions" that describe particles on a microscopic scale are all described by a family of closely related equations. When the media in which the waves propagate are rough or discontinuous, wave propagation is complicated by the effects of diffraction. This project is concerned with a number of aspects of wave propagation in settings where diffraction may play a novel role. The Principal Investigator (PI) will address fundamental questions such as: how do sound waves resonate in a medium where the sound speed may jump? how is a quantum particle affected by discontinuities in the underlying medium? how are internal waves in the ocean scattered by variation in the structure of the bottom? Potential areas of application include better design of quantum devices, improved control and damping of acoustic waves, and better understanding of the mixing of the ocean. The project provides research training opportunities for graduate students and postdoctoral scholars. The PI investigates questions involving wave equations in several settings with the goal of understanding the behavior of the wavefunctions arising in quantum mechanics when the potential function that governs the system may not vary smoothly. These results will also apply to the behavior of acoustic waves in settings where the sound-speed may not be smooth. Further, the PI studies the scattering of internal waves in the ocean as they propagate over a variable bottom topography. The effects on quantum scattering theory of diffraction from multiple Coulomb potentials are also analyzed. 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 2025 · 2025-08

PROJECT SUMMARY The follicle is the functional unit of the ovary composed of an oocyte surrounded by somatic support cells, including granulosa and theca cells. During folliculogenesis, bi-directional communication between the oocyte and the granulosa cells s accumulation of maternal RNAs, proteins, organelles, and other factors that sustain early developmental events prior to zygotic genome activation, including meiotic maturation, fertilization, and early preimplantation embryo development. Thus, folliculogenesis is an essential process. Over the past several decades, the development of ex vivo ovarian follicle culture techniques has provided a tightly controlled model system to study follicle-intrinsic processes independent of the ovarian microenvironment. There are many successful culture techniques, and one of the most frequently used methods across species involves encapsulation of follicles in alginate-based hydrogels which mimics the three-dimensional architecture of the follicle. These culture methods have revealed key details about the hormonal, molecular, and biomechanical factors important for folliculogenesis and ovulation. Despite these advancements, existing approaches of ex vivo follicle culture are technically demanding and are low throughput. One of the most critical challenges is that these systems produce a consistently low yield of high-quality eggs capable of supporting preimplantation embryo development. In this application, we will engineer a scaffold-free platform that better mimics ovarian biology with the aim to improve the developmental competence of the resulting gamete following ex vivo follicle culture. Through two specific aims, we will integrate and leverage a tunable agarose microwell design, timelapse imaging, and microfluidic culture to develop a follicle culture system capable of producing high-quality gametes. First, we will identify the ideal conditions for culture of high-quality follicles by engineering agarose molds that impart different biomechanical properties on the growing follicle and hone the timing of ovulation using growth parameters of individual follicles based on timelapse imaging. Second, we will use a microphysiological system to determine the effect of dynamic exchanges of fresh and conditioned media that better mimics in vivo nutrient delivery, hormone fluctuations, and waste disposal. The primary endpoints of the study will focus on the quality of the resulting eggs assessed by their ability to yield high-quality preimplantation embryos, which will be monitored by the Embryoscope™ time-lapse system. Our aims merge reproductive biology with innovative bioengineering techniques, which is an essential partnership for developing a biomimetic follicle culture platform that supports maximal egg and embryo quality. This research is expected to create a scalable and accessible tool for basic ovarian biology research and represents a necessary first step towards the clinical translation of this method in the context of fertility preservation.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Neural crest stem cells play a central role in vertebrates as they contribute numerous critical cell types to the body plan including much of the peripheral nervous system and craniofacial skeleton. Accordingly, understanding the molecular mechanisms controlling formation and development of these cells is key to understanding congenital defects, and for regenerative medicine. Pou family transcription factors, particularly the Pou5 and Pou3 subfamilies, play crucial roles in regulating pluripotency, differentiation, and various developmental processes across species. We have recently shown that Pou5 family factors are essential for neural crest formation. Evidence suggests that pou5 evolved from a pou3-like ancestor that was involved in neurogenesis, a function probably ancestral for chordates and therefore one that preceded the origin of pou5 function in the neural crest. The work proposed here will address the differences that have arisen in extant pou5 and pou3 proteins that underlie their distinct activities and targets. The proposed research will advance our understanding of neural crest cells, and provide a new framework with which to understand their origins and stem cell attributes, in addition to shedding light on the molecular underpinnings of a broad set of congenital defects. They will also serve as a paradigm for understanding the evolution of gene regulatory networks by duplication and divergence of key transcription factors.

NIH Research Projects · FY 2025 · 2025-08

Despite the central role for proteins as primary mediators of cell phenotype and function, proteomics has lagged genomics and transcriptomics as a tool for fundamental and translational biomedical research. There is a significant deficit in our knowledge of functional post-translationally modified forms of proteins, i.e. proteoforms, and the role of proteoform dynamics in health and disease. Technologies to readout proteoform biology are neither hardened nor easily adopted by the community at large. Despite an increasing number of first-wave commercial solutions over the last five years, the academic proteomics community has yet to integrate top down mass spectrometry approaches to proteomics into mainstream practice. We are proposing a new National Center for Translational and Developmental Proteomics (NCTDP) that will leverage the top down mass spectrometry methods developed over the past 9 years of NIGMS funding to optimize and disseminate cutting edge methods for proteoform discovery and measurement to the scientific community. The goals of the NCTDP are (1) achieve impactful outcomes for Driving Biomedical Projects; (2) reduce to practice selected technologies; and (3) and broaden access to new top down proteomics methods via robust dissemination across academic and industry research communities. These goals will be achieved using a four-pronged approach. First, we will optimize and beta test impactful workflows for proteoform measurement through three Technology Optimization Projects in preparation for dissemination and commercialization. Second, we will advance the mapping and robust measurements of proteoforms in basic and translational biomedical research through 10 Driving Biomedical Research Projects (DBPs). Third, the Center’s Community Engagement program will engage academic-, government-, and industry-based proteomics communities through on-site training, beta testing, and professional webinars. Implementation of NCTDP technologies as service lines in the well-established Northwestern Proteomics Core, plus hands on training for core directors from across the country, will enable broad access to Center technologies for external investigators and their students. And finally, we will provide robust leadership and supporting infrastructure that will facilitate integration of the technology optimization goals, effective execution of driving biomedical projects, and vigorous community engagement. The proposed NCTDP will harden the current generation of technologies for discovery and targeted measurement of proteoforms to bridge top down mass spectrometry with other -omics and single-molecule protein sequencing. The Center’s outreach and dissemination initiatives will address protein knowledge gaps and will significantly improve the capabilities of the research community to (a) better detect and assign function to proteoforms, their isoforms and PTMs, (b) create exportable assays to probe the early onset of cellular and disease phenotypes, and (c) determine new targets for more precise development of drugs and chemical probes. This expanded tool kit will usher in a new era of proteoform systems biology and biomedical research.

NIH Research Projects · FY 2025 · 2025-08

Summary Although it is becoming increasingly clear that CD8+ T cells responding to chronic infection are phenotypically and functionally diverse, little is known about how to overcome T cell exhaustion to treat infectious diseases. Using single cell RNA sequencing (scRNA-seq), the investigator’s team has confirmed the presence of previously identified TCF-1hi progenitor and PD-1hi exhausted T subsets. Surprisingly, they also found a CX3CR1+ effector subset at the late phase of chronic infection. More importantly, they have shown that CX3CR1+ effector cell formation is critically dependent on IL-21-producing CD4+ helper T cells. The discovery of this potent antiviral effector subset provides new opportunities for therapeutic interventions to treat infectious diseases. To better understand the genesis of CX3CR1+ effector cells, the investigator’s team proposes to use the newly developed technology of paired single-cell RNA and TCR sequencing (scTCR-seq) to “lineage trace” back to the ancestors (progenitors) of effector and exhausted cells. Their preliminary data suggest that TCR signaling strengths positively correlate with exhausted T cell subset differentiation, but negatively correlate with effector T cell differentiation. In parallel, they also provide evidence that CX3CR1+ effector cell formation depends on cross- presenting dendritic cells (DCs), especially Batf3+ XCR1+ cDC1s. These cDC1 cells are likely subjected to CD40- mediated CD4 helper T cell licensing, which is unexpected in the context of persistent infection and possibly occurs within specialized cellular structures. Taken together, the investigator hypothesizes that progenitor CD8+ T cells need to be primed and activated by the cDC1s again at the late phase of chronic infection to enter into a proliferative burst as a transitory differentiation state, and then bifurcate into two terminally differentiated subsets: CX3CR1+ effector and PD-1hi exhausted CD8+ T cells. Mechanistically, CD4+ T cells need to provide signals to license DCs for their antigen-presentation, and TCR signaling strength dictates the threshold of progenitor cell activation and influences the effector versus exhausted cell fate choice. Harnessing this knowledge, they intend to alter the course of T cell differentiation by changing TCR signaling strength through identification and validation of TCR signaling regulators (TSRs) that can be exploited to favor the formation of CX3CR1+ effector T cells for improved viral control. The investigator’s team will test these hypotheses in the following three aims. First, they will use paired scRNA- and scTCR- seq and associated computational tools and biological validations to delineate virus-specific CD8+ T cell differentiation trajectories and dissect how TCR signaling strength affects cell fate decisions. Second, they will use genetic models to delineate the mechanisms by which DCs are licensed by CD4+ helper T cells for CD8+ T cell priming and differentiation. Third, they will employ high-throughput targeted CRISPR screen and gene editing to test if manipulating TCR signaling strength can redirect CD8+ T cells toward effector differentiation and overcome exhaustion to treat chronic infection and cancer.

NIH Research Projects · FY 2026 · 2025-08

SUMMARY / ABSTRACT Bicuspid aortic valve (BAV) is the most common congenital heart defect (1-2% in the U.S.) which can lead to severe complications including aortic dilatation or dissection. However, the current risk-stratification paradigm is based on primitive aortic diameter thresholds and has poor predictive value for outcomes. Studies have shown that 4D flow MRI can measure in-vivo complex 3D blood flow as risk factors for aortic complications in BAV. At Northwestern University (NU), 4D flow MRI has been used for hemodynamic assessment of the aorta since 2011 and we have established a large database with over 4,000 aorta 4D flow MRI scans in >2,000 unique patients and >300 healthy controls. Using this data we have identified promising hemodynamic indices for risk stratification in BAV. For example, elevated WSS was found to be a novel biomarker for vessel wall architectural remodeling and identifies patients at risk of progressive aortic dilation. However, broad clinical adoption of 4D flow MRI is hindered by long scan times (8-15 minutes) and cumbersome analysis, limiting reproducibility and translation. To overcome these challenges, we developed deep learning (DL) models to highly accelerate 4D flow MRI acquisition and automate analysis workflows. Our goal is to build on these findings to establish an end-to-end 4D flow MRI DL pipeline for highly efficient data acquisition and hemodynamic analysis with full on-scanner deployment. The large NU database of manually processed 4D flow MRI data (n=4,000+) provides unique ground truth data for training and testing of DL workflows. To account for variability across sites and MRI systems, DL developments will be augmented by aorta 4D flow data by our subaward sites, chosen to allow for validation across the main MRI hardware vendors (NU: Siemens, UW: GE, CU: Phillips). The final DL networks and processing tasks will be embedded in containerized applications to allow for multi-vendor on-scanner deployment as well as dissemination and prospective multi-site evaluation. We aim to 1) rigorously optimize and validate deep learning accelerated 4D flow MRI for the accurate measurement of aortic hemodynamics; 2) utilize automated hemodynamic analysis to conduct a retrospective large cohort study with known 10-year BAV outcomes, and 3) conduct a prospective multi-center and multi- vendor evaluation study of the end-to-end 4D flow MRI deep learning pipeline. This study will introduce innovative DL methods for 4D flow MRI, assess the role of hemodynamics in BAV 10-year outcomes, and validate the multi-site feasibility and performance of the end-to-end 4D flow pipeline.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Critical to maximizing communication outcomes for children with developmental delays (DD) is access to a high dosage of high-quality early intervention as early in development as possible. Children with DD under 3 years of age qualify for early intervention (EI) services through publicly funded Part C programs under the Individuals with Disabilities Act. A central tenet of Part C is that EI services should occur in the child’s natural environment and include the family, rather than working exclusively with the child via therapist-delivered interventions. Such interventions are often referred to as “caregiver-mediated interventions” (CMIs). Several meta-analyses suggest that CMIs are effective for improving expressive and receptive language, social communication, and joint engagement. Caregiver coaching is a critical element of CMIs, yet despite strong empirical evidence, fewer than 25% of families receive caregiver coaching. Identified barriers to the widespread use of caregiver coaching include: (a) lack of training on caregiver coaching, (b) therapist and caregiver beliefs about their roles in early intervention, and (c) therapist confidence, skill, and self-efficacy in using coaching practices. These barriers and subsequent low rates of uptake highlight the disconnect between robust empirical support for caregiver coaching and implementation. Such a disconnect requires developing and testing implementation strategies that are tailored to address both: (a) the unique barriers to implementation in a given context (e.g., Illinois EI system), and (b) various components of caregiver coaching (e.g., observation, feedback). As such, the proposed study is guided by Implementation Mapping (IM), a 5-step participatory planning process to develop and test strategies that promote the uptake of caregiver coaching. Each of our study aims corresponds to a specific IM step and is informed by a specific implementation science framework. In Aim 1, we will select implementation outcomes that are guided by Proctor’s taxonomy of implementation outcomes. In Aim 2, we will use the Consolidated Framework for Implementation Research (CFIR) to characterize malleable factors that influence implementation outcomes. Specifically, we will analyze qualitative data from focus groups using a rapid deductive CFIR analysis approach. In Aim 3, we will integrate implementation outcomes with determinants to create a working Implementation Research Logic Model that maps implementation strategies to specific determinants. Potential implementation strategies will be selected using Expert Recommendations for Implementing Change (ERIC) and will be reviewed by EI therapists (n=15) and caregivers (n=15) during two, day-long, in-person workshops. In Aim 4, we will create the materials for the operationalized implementation strategies from Aim 3. Even strategies selected from the ERIC list require tailoring to EI and caregiver coaching. Implementation materials will be iteratively designed and refined using the Cognitive Walkthrough for Implementation Strategies. Results of this project will yield a fully developed set of co-created implementation strategies and materials that can be tested in a future hybrid Type 2 (effectiveness + implementation trial; R01).

NIH Research Projects · FY 2026 · 2025-08

The mission of the Northwestern Center for Reproductive Science Predoctoral Training Program in Reproductive Science, Medicine, and Technology (CRS Training Program) is to train future leaders in the reproductive sciences, while simultaneously improving human health. The CRS Training Program is embedded in the CRS, which has a four-decade history of keeping reproductive science and medicine visible, viable, and valuable. The CRS infrastructure provides a physical space for our trainees and an intellectual hub of data clubs, seminars, workshops, and summits. Over the first five-year duration of the program, our trainees have co-authored fifteen papers, including eight first author papers, presented at conferences and meetings in both oral and poster formats, received independent research grants, and received awards and recognitions for their outstanding predoctoral work. In the next funding cycle, we will continue to train predoctoral graduate students from five of Northwestern’s top graduate programs in the life sciences, bioengineering, and medicine. We will expand our course offerings, and we will enhance training outcomes by improving our training for mentors and continually adapting our program based on improved program evaluation mechanisms and trainee focus groups. Renewed funding of this program is critical as our freshly minted doctorates will enter the professional arena at an unusual time for science – one marked by low funding and fierce job competition. As such, our comprehensive educational program was designed with a focus on cutting edge and emerging technologies so we can develop successful leaders in reproductive science and across multiple disciplines in this lean, face-paced environment. Our trainees receive well-rounded training in reproductive science and medicine through coursework spanning didactics on basic and clinical reproductive physiology, reproductive technologies laboratory skills, research proposals and scientific communication, and responsible conduct of research. Our 21 Faculty Mentors come from 13 departments and study reproductive science and medicine from basic, translational, and clinical perspectives. Trainee coursework and laboratory training is bolstered by unique CRS Training Program experiences which focus on professional and career development experiences. All trainees participate in Academic Accelerator Partnerships, which are extended laboratory and core facility exchanges, or externships designed to expose students to team science and the latest technologies related to their research. Trainees also receive formalized professional and career development upskilling through our Career Catalyst Series which exposes our trainees to diverse career opportunities and prime them with career skills, including written, oral, and visual communication, self-assessment, networking, teamwork, and outreach. Ultimately, the CRS Training Program provides trainees with the most comprehensive formal training in our field.

NSF Awards · FY 2025 · 2025-08

Next generation spectrum access will require accommodating an ever-growing number of devices within the limited amounts of useable radio spectrum. Efficiently allocating spectrum to these devices is often expressed as an optimization problem which can be computationally very difficult to solve. This challenge is exacerbated when the external conditions (e.g., number of connected devices, presence of interference sources, and other parameters) change in real time, thus necessitating a fast solution to the spectrum allocation problem than can easily adapt to these changes. Existing software-based methods for spectrum allocation, which run on generic computing hardware, face significant difficulty in tackling such scenarios. We are developing custom-designed hardware in the form of application-specific integrated circuit (ASIC) chips, along with the appropriate algorithms, in order to accelerate the solution of these increasingly important optimization problems. This project is contributing to the continued U.S. leadership in wireless communications infrastructure, as well as its foundations in microelectronic hardware and advanced optimization algorithms. It also is contributing to U.S. workforce development, through outreach to K-12 students, involvement and training of graduate students, and integration of the research results into courses. This project is utilizing spectrum consumption models (SCMs) to enable devices to share their spectrum needs on finer spatial and temporal scales than possible today. Given this collected data, new algorithms for spectrum assignment are being developed that account for a rich set of priorities, aggregate interference, and temporal dynamics. Probabilistic computing hardware is being developed to accelerate the implementation of these algorithms and enable them to scale to a large number of devices. The hardware consists of programmable probabilistic computing solvers in the form of custom-designed ASICs, which are being fabricated through a commercial foundry. 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 2025 · 2025-08

PROJECT SUMMARY Dizziness is a common problem affecting nearly 37 million U.S. individuals annually and accounting for 2 million emergency department (ED) visits per year. Dizziness presents a substantial diagnostic challenge for clinicians because of its broad differential spanning both benign and serious diagnoses; dizziness-related language is also frequently used by patients to describe symptoms from non-balance related conditions. Thus, ED visits for dizziness commonly result in low value diagnostic testing (e.g., computed tomography, magnetic resonance imaging) and unnecessary hospital admission, which in turn contributes to escalating healthcare costs. Patients suffering from dizziness report reduced quality of life, anxiety, and depression, in addition to a substantial number of lost working days and time spent seeking healthcare. Fortunately, vestibular rehabilitation therapy delivered by physical therapists in the outpatient setting has been shown to improve dizziness symptoms for a number of balanced-related conditions. Whether vestibular rehabilitation therapy can be delivered by physical therapists in the emergency care setting – where patients have received little to no prior medical evaluation – is currently unknown but represents vast potential for benefit. This multi-site feasibility trial will evaluate the feasibility of emergency department vestibular rehabilitation therapy for patients with undifferentiated dizziness in two diverse health systems in Chicago, IL and Salt Lake City, UT. We will also gather qualitative data on contextual implementation determinants and preferred implementation strategies among stakeholders at multiple levels in preparation for a future multi-site hybrid effectiveness- implementation trial.

NIH Research Projects · FY 2025 · 2025-08

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurological disorders with limited treatment options. A hexanucleotide intronic (G4C2)n repeat expansion in C9orf72 is the most common genetic cause of ALS and FTD. However, patients with C9orf72 mutations are characterized by clinical heterogeneity and variable penetrance. The nature of this variability is not well understood but environmental factors have been proposed to act as an additional trigger beyond the pathophysiology associated with the C9orf72 repeat expansion. Moreover, 90% of ALS/FTD cases are classified as sporadic and likely arise from an interaction between genetic predisposition and environmental factors. One such environmental factor is neurotrauma, which results from traumatic brain or spinal cord injury and has been shown to substantially increase the risk of ALS/FTD in epidemiological studies. The association between traumatic injury and ALS/FTD is exemplified by the shared neuropathology TDP-43 aggregation. TDP-43 is a predominantly nuclear RNA binding protein involved in RNA transport and splicing, that forms cytoplasmic aggregations in more than 98% of ALS cases. Widespread TDP-43 aggregation is also seen in patients who have suffered traumatic brain injury (TBI). While these observations indicate a potential link between these diverse neurodegenerative diseases, it is unclear whether the mechanisms that drive TDP-43 pathology in ALS and TBI are shared or divergent. What also remains unclear is whether an ALS genetic background confers heightened vulnerability to neurotrauma. Here, we will use ALS/FTD patient-specific induced pluripotent stem cell (iPSC) derived neuronal cultures in combination with a custom-built device that applies biofidelic mechanical stretch-induced trauma in vitro, to establish novel human models that mimic the impact of traumatic injury. The innovative instrument can effectively recapitulate a key facet of TBI in diffuse axonal injury. In key preliminary work we found that C9orf72, but not isogenic control motor neurons exhibit selective degeneration and extended cytoplasmic accumulation of TDP- 43 in response to trauma. In Aim 1 we will interrogate several ALS C9orf72 iPSC lines and controls to test the hypothesis that stretch-trauma exacerbates C9orf72 pathology. We will measure both sense and antisense transcript and dipeptide repeat protein production in response to varying degrees of traumatic injury. We will investigate the ability of sense and antisense targeting ASOs to protect neurons from injury to dissect the relative contribution of these mechanisms to stress-induced mutant C9orf72 toxicity. In Aim 2 we will generate spinal and cortical neurons from 12 sporadic ALS and 12 age/gender matched control iPSC lines and interrogate their ability to cope with varying degrees of mechanical trauma. We will examine neuronal survival, the subcellular localization, solubility and functionality of TDP-43 protein using biochemical and RNA-Seq approaches. Our work will capture the interaction between mechanical trauma and an ALS/FTD genotype in a controlled environment that allows definitive hypothesis testing and mechanistic experiments

NIH Research Projects · FY 2025 · 2025-08

Project Summary Early electrical patterns in the human brain guide development, and even slightly disrupted electrical activity can result in severe neurodevelopmental diseases. Epilepsy alone affects ~3.5 million individuals in the United States, including ~450,000 children (CDC). For children with severe epilepsy syndromes, few effective treatments are available, and clinicians rely on decades-old anticonvulsant drugs. Recently, our lab, in collaboration with Tim Yu’s lab at Boston Children’s Hospital, obtained Food and Drug Administration approval for a precision-guided antisense oligonucleotide (ASO) RNA therapy that we developed and validated for a child who, due to potassium-channel malfunction, had a treatment-resistant epilepsy. This safe and efficacious RNA therapeutic modality (see also FDA approved Nusinersen for SMA) has transformed the therapeutic landscape as a cross-cutting approach for children with untreatable diseases. However, while ASOs are a proven therapeutic strategy, the “personalized precision medicine” model for individual patients costs millions of dollars per child, and is therefore both limited in its treatment reach and highly inequitable for low resource/underserved patient populations. Moreover, developing a personalized therapy takes an average of 3 years, by which time irreversible brain damage has occurred. Instead, this DP2 proposal aims to provide an immediately available and broadly applicable RNA therapeutic to stabilize children early with severe neurodevelopmental diseases. Here, we propose a transformational approach that will enable us to reprogram brain cells, enhancing their resilience against a broad array of neurodevelopmental diseases by stabilizing electrical activity before permanent damage can occur. To this end, we combine our proven RNA therapeutics approach to stabilize excitotoxicity pathways with a high-throughput 3-dimensional (3D) neurobiology platform optimized for the evaluation of complex neocortical neurophysiology in 100,000+ cerebral organoids over the project duration. If successful, our results will build a new era of stabilizing therapeutics for a wide range of diseases in infants. To support our HTS 3D cell culture and RNA-approach, we employ leading in silico modeling techniques, including machine learning, to simulate neuronal activity responses to ASOs. In vitro models will be screened against computationally designed ASO cocktail combinations to systematically modulate levels of neuronal activity in specific cell-types, monitored with dual physiological calcium and voltage readouts. We are committed to share our longitudinal single-cell and 3D-neurophysiology datasets with colleagues to enhance therapeutic targeting in human brain development and power the switch from traditional monolayer neuron models to 3D models that best capture complex human neocortex neurophysiology. This DP2 will reveal the full potential of this new class of ASO, improving the lives of children with neurodevelopmental diseases and pave the way for our highly efficacious RNA chemistry to be utilized for prenatal therapies (akin to in utero spina bifida surgery).

NSF Awards · FY 2025 · 2025-08

In this project funded by the Chemical Mechanism, Function, and Properties Program of the Chemistry Division, Professors Biwu Ma, Bin Ouyang of Florida State University and Professor Lin X. Chen of Northwestern University will investigate how light-induced structural changes in hybrid materials affect their optical and electronic behaviors. By combining material design, ultrafast optical and X-ray techniques, and theoretical modeling, the team will study an emerging class of organic-inorganic hybrid materials, organic metal halide hybrids (OMHHs), with deformable lattices. The project will provide insights to guide the development of next-generation light-responsive materials and devices. Participating graduate and undergraduate students will receive interdisciplinary training in synthesis, spectroscopy, and theory. The project’s findings will also support science education through outreach activities such as summer programs and public exhibitions. This project addresses fundamental questions about the coupling between electronic and atomic motions in photoactive materials, focusing on OMHHs with controllable 0D, 1D, and 2D structures. These low-dimensional systems offer an ideal platform to explore how structural reorganization under light excitation influences exciton dynamics and carrier transport. The research will combine three major strategies: (i) ultrafast optical spectroscopy to track charge carrier and exciton localization and dynamics on femtosecond timescales, (ii) ultrafast X-ray absorption and scattering techniques to reveal structural reorganizations at the atomic level, and (iii) density functional theory (DFT) calculations to model excited-state properties and structure–function relationships. The findings will advance fundamental knowledge of light-matter interactions and inform the rational design of next-generation hybrid materials with tunable optical and electronic properties. 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 2025 · 2025-08

ECR R21 – Project Summary The central auditory system modulates its responses to self-generated sounds that result from an individual’s movements. However, how the human auditory system balances two competing functions—coarse attenuation of self-generated sounds allowing us to remain vigilant to the external environment, and fine-grained accentuation of unexpected sounds to monitor for prediction errors—is not well-understood. We will use the frequency-following response (FFR)— which measures the fidelity of a sounds’ representation in the early auditory system—to assess the timing and location of auditory–motor interactions in the auditory system, whether subcortical or in primary auditory cortex. We will then uncover how early auditory processing of self-generated sounds is modulated when the auditory stimulus is unexpected and therefore does not align with sensory predictions. Separately, but in the same participants, we will use functional magnetic resonance imaging (fMRI) to identify cortical neural processing of auditory–motor interactions when hearing both expected and unexpected stimuli during active and passive sound presentation. Finally, we will directly compare early auditory FFR representations and later auditory cortical fMRI representations to map the neural processing of self-generated and expected sounds. By assaying auditory perception across self- vs. passive generation and expected vs. unexpected contexts, as well as by synthesizing non-invasive methods for imaging early and later auditory processing, this project will uncover fundamental information about how the human brain processes self-generated and passively perceived sounds, and how that processing changes when the sounds are unexpected vs. expected.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY Nucleotides serve as pivotal bioenergetic molecules vital for diverse cellular processes, encompassing RNA and DNA synthesis, glycosylation, phospholipid metabolism, energy production, signaling, and cytoskeleton function. During the past decade, my independent research has led to fundamental insights into the molecular mechanisms that govern the metabolic pathways, enabling cells to grow and proliferate. My laboratory has made significant discoveries by molecularly connecting the pro-proliferative signaling pathways to nucleotide metabolism. We have achieved landmark discoveries by precisely delineating the molecular regulation of de novo purine synthesis downstream of the ERK signaling pathway, elucidating the effects of mTORC1 signaling on the methionine cycle and RNA methylation, and demonstrating the pivotal role of environmental bicarbonate in fueling de novo nucleotide synthesis in response to mTORC1 activation. Recently, our findings have not only expanded our understanding of metabolic function but also reshaped the biochemical landscape. We have overturned conventional wisdom by revealing the indispensable role of pyrimidine nucleotides in supporting vitamin B1 metabolism and mitochondrial pyruvate oxidation and Krebs cycle function. These revelations hold profound implications, not only for our comprehension of metabolic physiology but also for the development of innovative drug strategies. The success of our efforts has been underpinned by the support of two NIGMS- funded R01 grants, serving as the lifeblood of our research pursuits. My lab has thrived in answering fundamental questions regarding cellular metabolism regulation and uncovering novel metabolic functions for purine and pyrimidine nucleotides. This proposal embodies the ethos of the MIRA funding scheme, empowering researchers to pursue ambitious science and delve into higher-risk endeavors. Through MIRA funding, we aim to uncover metabolic connections between the nucleotide synthesis pathways and metabolites, identify the molecular mechanisms of nucleotide sensing in human cells, and expand the horizons of our research endeavors. The stability and flexibility inherent in MIRA funding will enable us to propel scientific discovery forward, forging new knowledge in the realm of signaling and cellular metabolism and advancing our quest for innovations.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Membrane contact sites (MCSs) play critical roles in spatially organizing cells and facilitating the transfer of biological materials between organelles. MCSs are defined as sites of close apposition between membranes that are physically tethered by protein-protein or protein-lipid interactions and perform specific biological functions. These sites of contact are a ubiquitous mechanism used by cells to facilitate communication between organelles and are associated with a wide array of cellular functions. Given their importance in maintaining cellular homeostasis, it is not surprising that MCSs have been implicated in a wide range of diseases, including neurological diseases, cancer, and pathogen-induced diseases. Our plan for the next five years is to build on the successes we have had studying mitochondrial MCSs and address fundamental unanswered questions that are widely applicable to MCS biology. Our work will be primarily grounded in the yeast system. The simple organelle architecture of budding yeast has proven to be an excellent model for studying MCS form, function, and regulation in mechanistic detail. Our studies in yeast over the past five years have opened avenues of scientific exploration that have broadened the scope of our research program beyond a limited view of a single MCS. Our research program will follow three independent yet complementary directions, each of which will address a critical unanswered question in MCS biology. We will continue our studies on a tripartite membrane contact site between the mitochondria, endoplasmic reticulum, and plasma membrane to elucidate how and why three distinct membranes are brought into functional close contact. We will explore the fundamental mechanisms by which MCSs are coordinately regulated at two different length scales— within a shared space and across the MCS network. We will also examine the role of lipids as critical structural, functional, and regulatory components of MCSs. Our prior accomplishments and plans for the next five years will place us in a strong position to initiate studies in mammalian systems, in which we will examine how the fundamental principles of MCS formation, function, and regulation we uncover in yeast are utilized in more complex cellular settings. Our goal is to uncover fundamental mechanisms used by cells to form and regulate interorganelle contacts and deepen our understanding of MCS physiology. In doing so, we anticipate this work will provide insight into novel therapeutic strategies for a range of human disease conditions in which the manipulation of MCSs can be used to positively influence cellular health and homeostasis.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Rising antibiotic resistance and the emergence of multi-drug resistant pathogens requires the discovery of new drug targets and therapeutics. Currently, the most prominent source of new medicines is natural products, or secondary metabolites. The proposed research involves the investigation of biosynthetic gene clusters (BGCs) relevant to pathogenesis and utilizes genome mining of natural product BGCs containing metal-dependent enzymes as a query to uncover natural products with novel chemical structures. Metalloenzymes are responsible for many unusual chemical reactions in natural product biosynthesis. We recently identified a copper-binding natural product from nontypeable Haemophilus influenzae (NTHi), which we termed oxazolin. Since oxazolin is required for NTHi to infect human cells, its biosynthesis may serve as an ideal drug target. The core enzyme in oxazolin biosynthesis is a multinuclear non- heme iron-dependent oxidase (MNIO), which belongs to a poorly understood family of enzymes that produce ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. We hypothesize that oxazolin serves a protective role for NTHi, chelating toxic levels of copper that human cells employ to stave off infecting pathogens. Aim 1 (K99 Phase) of the proposed research involves structural and functional characterization of copper-bound oxazolin and investigation of another oxazolin from the increasingly multi-drug resistant pathogen Pseudomonas aeruginosa. The latter half of this proposal, which will take place during the R00 Phase, builds on initial genome mining for metalloenzyme-containing RiPP BGCs to uncover novel chemical transformations in natural product biosynthesis. Genome-mining for other MNIOs revealed an operon from Streptomyces laurentii encoding two MNIOs and a secreted natural product; Aim 2 involves determining the function of each enzyme in this BGC and the structure of the natural product it produces. At least one of the MNIOs is predicted to carry out a new chemical reaction for this family of enzymes. A second growing family of iron-dependent enzymes in natural product biosynthesis is the heme oxygenase-like dimetal oxidases (HDOs). Mining for RiPP BGCs with HDOs identified an operon in Paraburkholderia diazotrophica encoding three HDOs of unknown function and an efflux pump, which likely facilitates secretion of the final product, perhaps to serve as an antimicrobial. Aim 3 includes characterization of each enzyme in the biosynthetic pathway and structure-function investigation of the HDOs. Not only will this line of inquiry reveal new natural products, but it will also provide biochemical insight into two families of iron-dependent enzymes with high biosynthetic value.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Developmental and epileptic encephalopathies (DEEs) are a group of severe disorders characterized by developmental delay/intellectual disability (DD/ID), infant-onset seizures, electroencephalographic (EEG) abnormalities, and elevated mortality risk. DEEs are primarily due to monogenic variants that arise de novo in the affected child. Although each gene-based etiology is rare, collectively DEEs are estimated at 1 in 2,000 live births and represent a significant public health burden due to lifelong disability. Poor response to available treatments is characteristic of DEEs; thus, there is a significant unmet need for effective therapies. De novo pathogenic variants in genes involved in neurotransmission are a leading cause of DEEs. A growing number of voltage-gated potassium channels have been implicated, including KCNB1 that we first reported as a DEE gene in 2014. Since then, at least 150 cases have been confirmed, and over 400 variants in KCNB1 have been identified via clinical genetic testing. Additionally, the clinical spectrum has expanded. Currently, KCNB1 encephalopathy includes DD/ID accompanied by behavioral and movement disturbances, and epilepsies of varying severity, ranging from infrequent seizures to frequent seizures poorly controlled with available treatments. KCNB1 encodes the alpha subunit of the Kv2.1 voltage-gated potassium channel, which has both classical ion conducting activity and non-canonical function at ER- plasma membrane junctions (EPJ). Kv2.1 classical ion conducting activity is critical for neuronal membrane repolarization during action potential trains. Non-canonical activity occurs at EPJ in neurons where dense Kv2.1 clusters serve as non-conducting signaling hubs and facilitate activity-dependent calcium signaling that is critical for synaptic transmission. Classical and non-canonical activities of Kv2.1 contribute to neuronal excitability; thus, functional characterization of variants should assess both. Yet, functional studies to date focused only on classical ion channel activity and protein expression. Thus far, ~30 variants have been characterized with most having loss-of-function effects. However, our current knowledge is incomplete as surveyed variants were biased toward severe DEE, and clustering and EPJ function have been unexplored. We hypothesize that KCNB1 encephalopathy encompasses a range of functional defects on conducting and/or non-conducting functions. To address our hypothesis, we will: (1) determine whether KCNB1 variants are stably expressed at the plasma membrane using immunocytochemistry/flow cytometry; (2) determine how KCNB1 variants impact classical ion conducting function using high-throughput electrophysiology; and (3) determine the impact of KCNB1 variants on non-canonical function by assessing clustering and ER calcium signaling. Our proposed studies will reveal the spectrum of pathogenic mechanisms by evaluating >450 KCNB1 variants, which will provide important functional evidence to aid clinical genetic interpretation and promote development of mechanism-targeted therapeutic interventions.