Columbia University Health Sciences

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT In the last few decades, novel pathogens have emerged at an unprecedented rate, causing significant health and economic burdens in our society. However, our ability to detect and quantify key epidemiological features of these pathogens has been limited during the early phase of outbreaks – a critical but short time window to understand new diseases. This gap arises primarily due to a lack of observational data and incomplete detection of transmission events, constrained by the resources invested in traditional surveillance systems. Our research group has been at the forefront of developing mathematical models and computational tools to advance the methodology for surveillance, inference, and forecasting of emerging infectious agents, including SARS-CoV-2, pandemic influenza, and antimicrobial-resistant organisms (AMROs). Our goals for the next five years are to develop novel computational tools to detect early signals of cryptic transmission and infer critical epidemiological features in data-sparse settings. Leveraging theories in complex systems and advanced techniques in machine learning and deep learning (ML/DL), we propose to synergistically use traditional (e.g., syndromic surveillance, PCR tests, sequencing, human mobility, and contact tracing) and non-traditional (e.g., wastewater, social media, and search queries) data sources to conduct a series of studies centering on three key questions. 1). How can we detect early transmission of emerging infectious diseases? We will take a biomimicry approach, inspired by the mechanism of the human physical sensation system to detect external stimuli (e.g., light, sound, and pressure) spanning several orders of magnitude in intensity. We will develop and optimize excitable sensor networks that collectively assimilate a multitude of data sources in different locations to assess the transmission potential for novel pathogens. 2). How can we infer key epidemiological features of novel pathogens using limited data? We will combine process-based models (e.g., metapopulation and agent- based models) and ML/DL techniques (e.g., Graphical Neural Networks and Transformers) to identify signatures of disease characteristics, such as asymptomatic shedding and superspreading, using early-stage data. 3). How can we validate model-derived hypotheses to reduce uncertainty? Determining characteristics of novel pathogens is a high-stakes task. We will develop strategies (e.g., sampling in specific locations at certain times) to cost-effectively validate hypotheses on spatial spread of new viruses and gather new evidence in real time to reduce uncertainty. For all three projects, the developed methods will be applied to a range of infectious agents with disparate epidemiology, including SARS-CoV-2, influenza, AMROs, and zoonotic viruses. We will apply new methods to retrospectively acquired data and quantify their advantage over classical approaches (e.g., how much data is needed with classical versus new methods). Our vision of the research program is that the synergistic use of computational tools and diverse data sources can enhance our ability to perform timely and impactful research to understand novel pathogens using sparse and imperfect data.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT To ensure accurate perception of the world, nervous systems must distinguish between sensory inputs caused by external stimuli and those generated by the body’s own movements. During rapid eye movements, or saccades, the brain suppresses sensory signals from self-generated motion through a process known as saccadic suppression. However, little is known about how this suppression affects the feature encoding capabilities of specific cell types within a visual network and the impact on broader visual processing. This project aims to elucidate the neural mechanisms of saccadic suppression using the Drosophila melanogaster model, which offers unparalleled genetic access and a tractable nervous system with a fully mapped connectome. The research will focus on the Lobula Columnar (LC) cell network, which encodes specific visual features and is known to exhibit suppression during body saccades. The project has three specific aims: (1) to characterize the neural inputs contributing to saccadic suppression in LC neurons through electrophysiological recordings and connectomic analysis, (2) to evaluate how saccadic suppression affects feature encoding across the LC network using population-level two-photon calcium imaging and advanced analytical techniques, and (3) to develop a computational model predicting how suppression impacts distinct LC subpopulations and their contributions to visual perception. This research will provide fundamental insights into how self-generated motion is processed and suppressed across visual systems. As part of the fellowship, I will receive training in electrophysiology, calcium imaging, and computational modeling under the mentorship of Drs. Gwyneth Card, Rudy Behnia, and Larry Abbott at Columbia University’s Zuckerman Institute. This world-class environment will provide me with the necessary skills and resources to achieve my long-term goal of establishing an independent research program focused on sensorimotor integration and neural circuit function.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease characterized by extreme chemotherapeutic resistance and the lack of effective immune response, fueling the need for effective treatment strategies. PDAC features include a complex desmoplastic stroma comprising cancer-associated fibroblasts (CAFs), a variety of lymphoid and myeloid cell lineages, and a plethora of paracrine signals that communicate between them. Malignant cells attract and reprogram stromal cells through morphogens like Sonic Hedgehog (SHH) and Transforming Growth Factor beta (TGFβ), creating a hospitable tumor microenvironment (TME) conducive to survival, growth, invasion, and escape from immune response. However, the underlying mechanisms mediating stromal reprogramming are understudied. This proposal aims to identify how active morphogen signaling shapes local immunosuppression in the pancreatic TME by focusing on the interaction of malignant epithelial cells, CAFs, myeloid cells, and T cells. In the past, targeting either of these populations has been insufficient to reverse immunosuppression, demonstrating that the complexity of crosstalk between diverse cell types in PDAC is insufficiently understood. By combining in vivo and ex vivo studies with a novel PDAC tumor explant platform, we are able to study cell-cell communication in an intact TME by manipulating morphogen signaling through targeted drugs, recombinant proteins, and neutralizing antibodies. These experimental approaches are guided by sophisticated computational analyses based on single cell RNA sequencing data. The overarching goal is to understand how CAFs and myeloid cells collaborate to maintain local immunosuppression within the pancreatic TME and leverage this understanding to reactivate anti-tumor immune response in PDAC. I hypothesize that combinatorial CAF and myeloid targeting will disrupt local immunosuppression and lead to partial reinstatement of T cell action that can be pushed further by immunomodulators. I will pursue this hypothesis through the following interrelated Specific Aims: In Aim 1, I will focus on how SHH-activated CAFs regulate the expansion of the myeloid cell population in PDAC. Aim 2 centers around building and testing a novel treatment regimen to reactivate exhausted T cells. Aim 3 seeks to identify differences between SHH and TGFβ-mediated CAF activation and downstream paracrine effects in the pancreatic TME. These studies will identify how malignant epithelial cells, CAFs, and myeloid cells work together to maintain immunosuppression through an intricate communication network. Successful completion of this proposal will both expand our basic understanding of PDAC immunosuppression and also produce a practical advance in the therapeutic targeting of this devastating disease. Concurrently, this work will facilitate my pathway towards an independent NIH-funded tenure-track faculty investigator by advancing my long-term objective of dissecting paracrine signaling cascades in PDAC.

NIH Research Projects · FY 2024 · 2025-09

Project Summary/Abstract In the U.S, preterm birth affects 1 in 10 pregnancies, and about half of preterm births are spontaneous as opposed to clinically-indicated inductions or cesarean sections. Despite the grave and widespread threat posed by spontaneous preterm birth (sPTB), its etiology is not fully understood, and diagnostic and therapeutic tools remain limited. Research has independently linked human genetics and vaginal microbes to preterm birth, but no study has explored links between all three entities. Furthermore, research on the associations between genetics and the vaginal microbiome has been constrained by small sample sizes, heterogeneous sample populations, and the use of 16S rRNA sequencing, which cannot classify bacterial taxa at the subspecies level. To define sPTB risk factors and develop effective diagnostics and therapeutics, we must gain a high-resolution understanding of microbial and genetic contributions to prematurity, including causal effects. The objective of this proposal is to investigate associations between host genetics, the vaginal microbiome, and sPTB in a large, diverse cohort with metagenomic sequencing. The nuMoM2b study collected genotyping, vaginal swabs, and extensive clinical data on >10,000 women from eight sites across the U.S. I will detect sample processing errors in this cohort by comparing genotypes inferred from metagenomic sequencing of vaginal swabs to those independently obtained in chip genotyping. I will generalize this method such that it may be applied to any study with both genotyping and metagenomic sequencing. After using my method for quality control in the nuMoM2b cohort, I will conduct Genome Wide Association Studies to detect genetic variants associated with the microbiome. I will investigate variants associated with microbiome characteristics imperceptible by 16S sequencing, such as the relative abundances of bacterial strains or their genomic profiles. I will then utilize microbiome-associated genetic variants in Mendelian Randomization analysis to probe causation of the microbiome on sPTB. Finally, I will train a predictive model on both genetics and microbiome data and benchmark it against models trained on either data source alone. This project will investigate causal effects of microbiome characteristics on sPTB, thus elucidating sPTB’s etiology and providing targets for novel interventions. The machine learning model will demonstrate the potential of diagnostic tools that predict sPTB risk from vaginal microbiota and genetics, which can be easily collected via vaginal swabs and blood testing, respectively. At Columbia, I have access to the facilities, equipment, and mentorship necessary to complete the proposed work. The F31 Ruth L. Kirschstein NRSA will support completion of this specific project while broadly encouraging my academic and professional development, including my progress towards a career as an independent investigator using computational tools for the study of reproductive biology.

NIH Research Projects · FY 2025 · 2025-09

Project Summary The primate visual system must balance competing demands, including representing objects and places in a format that is both abstract enough to be recognizable under novel conditions, but also specifically bound to other objects and places via contextual associations. How the visual system achieves this balance remains to be understood, as current artificial neural network (ANN) models fail to exhibit this same robustness and flexibility. One notable difference between ANNs and the brain is that the former preponderantly model the visual system as a single feedforward hierarchy, while ample evidence suggests that the latter in reality includes numerous parallel hierarchies with possibly complementary propensities for abstraction versus granularity. Aim 1 addresses such variations in representational geometry over cortical space by contrasting joint encoding of multiple objects in inferotemporal cortex (IT) and parahippocampal cortex (PHC) of common marmoset monkeys. I hypothesize that representations of different objects in IT will be comparatively independent of each other, while PHC will more nonlinearly encode groups of objects. Artificial neural networks trained on various loss functions, architectures, and training diets will then be used to attempt to model these phenomena. Aim 2 will investigate variation in representational geometry over the course of familiarization with a novel virtual environment over several days. I hypothesize that neural representations of different views of the learned environment will acquire the same correlation structure as the images themselves in the underlying latent factor space. This research will suggest new, non-standard neural network architectures for modeling vision, help us make more precise predictions about the effects of stroke or lesion on different parts of the brain, and better understand the underpinnings of disorders like attention deficit hyperactivity disorder, autism, frontotemporal dementia, and face and object agnosias. The training plan described in this proposal will afford me proficiency in new experimental and theoretical methodologies, including large-scale, multi-area electrophysiological recording, the use of common marmosets as a model organism for visual neuroscience, deep neural network modeling, and advanced data analysis techniques for understanding the structure of neural representations. I will receive expert training in electrophysiology and machine learning from my sponsor Dr. Elias Issa, as well as in theoretical neuroscience and advanced neural data analysis methods from my co-sponsor Dr. Stefano Fusi. This work will be conducted at Columbia University’s Zuckerman Institute, a world-class neuroscience research and training institution whose faculty specialize in approaches ranging from molecular to systems to theoretical.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Stillbirth is far too common in the U.S. with 20,000 stillborn babies every year with large racial and ethnic disparities. Black birthing individuals have twice the risk of stillbirth compared to White individuals. The Collaborative Action for Research to End Stillbirth Research Center (CARES) will conduct innovative, integrated, multilayered research - with and for people with lived experience of stillbirth squarely at the center- to develop and implement actionable strategies to improve the prediction of stillbirth early in pregnancy and to understand reasons for the excess risk for Black birthing individuals. We propose to address a critical area in stillbirth care – early identification of patients at highest risk for stillbirth to facilitate prevention. Screening patients early in pregnancy is important as 63% of stillbirths occur before 32 weeks of gestation. Joining efforts from researchers across essential disciplines, people with lived experience of stillbirth, and community, public health, and health care stakeholders, CARES will address the following Specific Aims: Aim 1. Establish a strong infrastructure to support stillbirth research and collaboration across the consortium. With input from the CARES Stakeholder Advisory Board and the CARES Parent Advisory Board, we will develop and maintain a core team of investigators, collaborators and staff with expertise essential for a broad range of stillbirth-related research. We will also build shared resources to promote interdisciplinary team science and ensure access to a diverse patient population to facilitate stillbirth research. Aim 2. Develop a scalable, electronic health record (EHR)-based, dynamic risk prediction model for stillbirth. Inadequate data sources have been identified as a major barrier to advancing knowledge on stillbirth and its prevention. We will (2a) Ascertain patients’ and health care professionals’ perspectives on risk factors for stillbirth (overall and for Black birthing individuals specifically), data needs for stillbirth research, and the acceptability and design of a mobile app-based patient data reporting tool to support pregnancy monitoring and stillbirth prediction; (2b) Develop and validate EHR-based and artificial intelligence-powered algorithms for automated identification and characterization of stillbirth, measurement of stillbirth risk factors, and prediction of stillbirth risk scalable across institutions; (2c) Develop and test a mobile app for patient-reporting of pregnancy information to augment EHR data and support pregnancy care. Aim 3. Develop novel markers of underlying incipient placental dysfunction early in pregnancy for predicting stillbirth. Fetal growth restriction (FGR) is a major antecedent to stillbirth with the most common underlying pathogenesis being placental dysfunction. Accurate identification early in pregnancy and management of FGR could potentially make an impact on stillbirth prevention. We will conduct a prospective cohort study to investigate innovative (3a) placental imaging before 14 weeks’ gestation, (3b) placental biomarkers, and (3c) fetal genetic markers for predicting fetuses at greatest risk for stillbirth. CARES will use multifaceted approaches applicable to the entire population of birthing people to prevent stillbirth.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Significance. Recent national drug-involved overdose death data has shown that the current opioid epidemic is characterized by four salient waves that claimed over 106,000 lives in 2021 alone. The current fourth wave of the epidemic is characterized as an opioid-stimulant polysubstance use. However, it is still unclear how individuals transition through different substance use profiles, the individual and structural determinants that are associated with these transitions, and polysubstance use profiles that are at increased risk of overdose. Additionally, no study to date has used a multilevel approach to develop a machine learning risk prediction model to predict overdose risk. Career Development Plan. Dr. Cadet’s training program will include seminars, workshops, coursework, and conferences to develop her skills and expertise in Multilevel Latent Markov Modeling, Bayesian Inference, longitudinal causal modeling, and in machine learning risk prediction modeling, which are necessary for conducting her proposed research plan and achieving her career goals of becoming an independent substance use epidemiology scientist who conducts large observational studies that will inform the targeted public health intervention and polysubstance use related overdose prevention. Mentorship. A highly accomplished team of mentors (Drs. Martins, Musci, Stingone, Tabb, Aiello) who are experts in substance use epidemiology, Bayesian Inference, longitudinal structural equation modeling, biostatistics, and machine learning, will support Dr. Cadet’s research and training goals. Research Plan. Dr. Cadet will conduct a multilevel epidemiological study informed by the Risk Environment Model that leverages the size and scope of the National Longitudinal Study of Adolescent to Adult Health (Add Health) from 1994-2026 (N> 20,000 participants) to answer the following aims: 1) Examine the dynamic transitions across substance use patterns using Multilevel Latent Markov Models (Latent Transition Analysis) among people who used drugs enrolled in the Add Health study; 2) Examine whether polysubstance use typologies have poorer survival risk of fatal overdose by using latent class modeling with a time-to-event distal outcome joint-model approach to explore latent polysubstance use classes with higher risk of fatal overdose and all-cause mortality in the Add Health study; 3) Develop a multilevel machine learning algorithms using a train-test split procedure to predict people who use drugs (PWUD) who are at increased risk of overdose episode by using micro-level (e.g., behavioral, psychosocial) and macro-level (e.g., area deprivation) factors in the Add Health study. Findings from this study will inform an R01 to lead a mixed-methods approach to better understand the nuances between intentional polysubstance use and treatment implications. Public Health Impact. The cross-disciplinary methodologies such as combining structural equation modeling, Bayesian modeling, and data science is crucial for addressing the multifaceted challenges of polysubstance use effectively.

NIH Research Projects · FY 2025 · 2025-09

The International Workshop on Pediatrics & HIV is organized on an annual basis prior to the biennially organized IAS or AIDS Conference. This workshop is the only meeting entirely devoted to research in prevention and treatment of HIV infections in infants, children, adolescents and pregnant and breastfeeding women, making it the primary forum for the world’s leading researchers. By bringing together experts from different disciplines with presentations in a variety of formats, the meeting offers a collaborative setting where the latest developments are presented, discussed, interrogated, and evaluated. The 17th Workshop is scheduled to be held in Kigali, Rwanda and virtually on 11-12 July 2025, prior to the 13th IAS Conference on HIV Science (IAS 2025). The program will include topics from among the following: Antiretroviral Treatment in Infants, Children, and Adolescents; Clinical Management of Infants, Children, and Pregnant/Breastfeeding Women; Coinfections/Complications in Infants, Children, Adolescents, and Pregnant/Breastfeeding Women with HIV; Clinical Issues in HIV Negative Infants Exposed to HIV; Prevention of Vertical HIV Transmission – Interventions and Implementation; HIV Prevention & Treatment in Pregnant and Breastfeeding Women; Clinical Management Issues Specific to Adolescents; HIV (and STI) Prevention in Adolescents; and Pediatric HIV Case Finding Including Early Infant Diagnosis. Given previous success with this format, we expect the 18th and 19th editions to follow this same pattern, including a similar program and schedule. The meeting format is also highly innovative. Moving from a more traditional format of plenaries and oral abstract sessions, the organizers have introduced unique approaches including structured debates, oral poster presentations, poster walks, clinical-case presentations, curated panel discussions, video presentations and a social program including a networking dinner where early career investigators meet with senior researchers. Attendance has increased substantially in the last several years with 292 delegates in 2024; highly favorable annual evaluations underscore the Workshop’s importance. The aims of the workshop are to 1) provide a platform for presentation and discussion of the latest developments in the field; 2) gather leading researchers involved in pediatric and perinatal HIV in a stimulating, interactive forum; and 3) promote the next generation of researchers. The objective of this proposal is to provide support for three annual workshops including participation of plenary speakers as well as members from the community of adolescents with HIV infection, and to increase the number of scholarships for early career investigators.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Youth living with HIV (YLWH) have high rates of mental health problems, and poor mental health is associated with worse HIV care continuum outcomes. Though the evidence for mental health interventions effective at improving mental health and HIV outcomes among YLWH is growing, data on whether they can be feasibly and cost-effectively implemented is lacking, and integration of mental health care into routine HIV programming remains rare. Mozambique, a low-income country, has the second highest number of new HIV infections in Eastern and Southern Africa, and just 54% of YLWH there are currently on antiretroviral therapy (ART). Recognizing the importance of mental health to optimal HIV care continuum outcomes, the Mozambican Ministry of Health has begun scaling up the Common Elements Treatment Approach (CETA) for people living with HIV, delivered by psychologists within the National Health System. CETA is a modular, transdiagnostic intervention developed for use in low- and middle-income countries, with demonstrated effectiveness in treating common mental disorders (CMD, i.e., depression, anxiety, PTSD) in Mozambican YLWH. However, like other high HIV- burden contexts, the prevalence of CMD among Mozambican YLWH (~10-20%) exceeds the resources available to deliver this already streamlined, individual intervention to all those needing care. To reduce the treatment gap, there is an urgent need for effective, more efficient mental health treatment options for YLWH. Leveraging our team’s research-service-policy partnership, we propose testing a novel, stepped-care model to streamline mental health services for YLWH within the Mozambican National Health System. We aim to integrate Interpersonal Psychotherapy-Adolescent Skills Training, an evidence-based intervention for youth experiencing mild-moderate CMD, within existing peer-led HIV support groups to develop an evidence-based, low-intensity intervention that simultaneously promotes mental health and HIV outcomes, i.e., CombinADO-MH (Aim 1). We will then conduct a Hybrid Type I Effectiveness-Implementation cluster randomized control trial to evaluate the clinical and implementation outcomes of delivering stepped care (CombinADO-MH for YLWH with mild-moderate CMD symptoms, CETA for YLWH with severe CMD symptoms) compared to CETA alone (all YLWH with mild-severe CMD symptoms). We hypothesize that CombinADO-MH will perform similarly to CETA at reducing CMD symptoms, increasing ART adherence, and increasing viral load suppression (Aim 2) but will have a greater reach and be a more cost-effective approach (Aim 3), producing greater overall benefits to YLWH. Addressing NIMH Strategic Goals 3 and 4, this pragmatic trial will provide data on the effectiveness, implementation, and cost of an innovative stepped approach to delivering evidence-based mental health care using peer-providers and integrated within HIV support groups, which may efficiently and synergistically improve mental health and HIV care continuum outcomes. Results can directly inform mental health and HIV programming in Mozambique and may serve as a model for other resource-limited contexts.

NIH Research Projects · FY 2025 · 2025-08

The need for improved radiation effect mitigators stems from the significant probability of the detonation of an Improvised Nuclear Device (IND). Typically pharmaceutical radiation countermeasures have been tested against x rays delivered over a few minutes. In practice, however, 1) the majority of the IND-related radiation dose will be delivered by “direct” radiation from the IND, over a time period of less than a microsecond, and 2) A significant component (10 - 50%) of the biological effect from a ground-burst IND will be from fission neutrons. The biological effects and associated mechanisms from these IND-specific radiations are typically quite different as compared with the corresponding effects and mechanisms associated with conventional x rays delivered over a few minutes. In addition there is evidence that mitigator performance is different for these IND- specific radiation types. It is therefore important to understand whether radiation countermeasures that have been developed and tested against x rays delivered over time periods of a few minutes will also be effective in these more realistic IND exposure scenarios. At Columbia University unique radiation sources are available to simulate each of these IND-specific exposures. These tools will allow each of the Research Projects to address the same common research themes: 1. Are radiation mitigators that were developed and tested with x rays irradiated over a few minutes, also effective against a) very short, very high dose rate radiation exposures, b) IND-specific neutrons? 2. Using these realistic IND exposures, can mitigators developed for specific endpoints have utility for other key radiobiological endpoints? 3. Using these realistic IND exposures, can combining multiple mitigators that were each designed for specific endpoints, have increased overall utility if given in combination (“polypharmacy”)? In this highly synergistic proposal, the Projects and Cores are tightly linked by common research themes and hypotheses, by a shared animal model, and by shared samples and data. The WAG/RijCmcr rats that will be used sequentially manifest our radiation endpoints of interest, allowing the sequential study in the same irradiated animals of GI-ARS (Project 1), lung and kidney damage (Project 2) and cardiovascular damage (Project 3). Each Project also features state-of-the-art pharmaceutical mitigators that have already been designed and tested by the relevant project team, using conventional x rays for their respective endpoint. The Proposal also features three strong central Research Cores (Radiation, Biomarker and Statistics), unifying the Research Program. The 7 Project and Core Teams already have a strong track record of working together, with >260 Team-Team interactions in publications, and >50 Team-Team interactions in grants - largely from NIAID RNCP grants. Finally a highly experienced Administrative Core will provide fiscal management support and will organize an annual 1.5-day retreat, coupled with a full-day training session targeting junior radiation scientists.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY A fundamental question in brain development is how a small group of neural progenitors use a single genome template to generate the vast diversity of neurons that underlie cognitive and motor function. It is generally thought that the three dimensional packaging of the genome underlies cell type specific gene expression. Thus, the physical organization of genes within nuclei must be specific to cell type and developmental stage. While much progress has been made with high throughput approaches to uncovering genome-wide organizational principles at different scales, what level of organization relates to gene function remains largely unknown. These questions are particularly challenging for neural progenitors, which can produce distinct neuronal subtypes over time. First, obtaining cell type and stage-specific genome profiling data that would allow one to detect meaningful organizational features is technically difficult, and such features would be lost by averaging among bulk heterogeneous cell populations. Second, there is a dearth of known trans-acting regulators of genome organization that can be used for gain/loss of function studies to test their impact on genome organization. Third, there are few experimental platforms in which to manipulate genome organization and validate their functional significance on downstream gene expression in vivo. Here, we have established the Drosophila neuroblast (NB, fly neural progenitors) system as an in vivo model to investigate how neural progenitor genome organization impacts its ability to produce distinct transcription programs in the descendent neuronal progeny. We previously showed that the hunchback (hb) gene, a key molecular signature of early-born neurons, relocates to the NB nuclear periphery, where it becomes heritably silenced and refractory to activation in the postmitotic neurons. Thus, changes in the genome organization of the NB progenitor directly regulates competence of the postmitotic progeny to transcribe hb. We discovered 250bp cis-acting element with the hb gene that is necessary and sufficient for hb gene relocation to the NB nuclear lamina, and mapped similar sequences genome-wide. Further, we have identified a trans-acting factor that regulates hb gene relocation and NB competence via its liquid-like, condensate forming properties. Here we will combine genetic and high throughput studies on purified, stage- specific NBs to test how genome dynamics are regulated in neural progenitors in vivo. We will then test the transcriptional consequence of genome reorganization of the NB on gene expression of the descendent neuron. Together, our proposed studies will provide mechanistic insights into how genome organization is regulated in neural progenitors and how genome dynamics contributes to neuronal diversification.

NIH Research Projects · FY 2025 · 2025-08

Over the past few years, focused ultrasound (FUS) has emerged as a promising noninvasive approach capable of both stimulating and suppressing neuronal activity. Ultrasound has several advantages over the aforementioned technologies as it can penetrate the brain over several centimeters through the intact scalp and skull. Given its entirely noninvasive and nonionizing nature, the technique has been shown to be translatable to human brain studies without requiring introduction of electrodes or optical fibers. Similarly, FUS has been applied noninvasively on peripheral nerves providing similar advantages. FUS works by modulating the neuronal tissue region where the FUS beam is focused and could be combined with readouts that inform level of activation. Several studies have shown that FUS can stimulate and/or suppress electrical and/or hemodynamic activity in rodents, non-human primates (NHP) and humans, induce limb movement, pupil dilation as well as suppression of somatosensory evoked potentials (SSEP) in mice evoke anti-saccades in NHP and suppress pain sensation in humans. In sharp contrast with its premise, however, and despite the multitude of advantages, FUS remains severely limited due to the lack of intra-animal reproducibility, lack of targeting and monitoring methodologies during modulation and its unknown underlying mechanism or targeted region. The true potential of FUS remains diminished in its expedited adoption and therefore risks faltering on its premise. To this purpose, our group has developed novel ultrasound-guided (USgFUS) capabilities with feasibility shown in both CNS and PNS FUS modulation. In the proposed studies, we aim to harness those methodologies developed to target regions critical for pain treatment. The methodologies proposed could thus constitute breakthroughs in FUS modulation since they allow to selectively focus (on the order of a few millimeters) and apply in shallow and deep-seated regions while informing on the type of FUS mechanism in real time.. In the proposed R18 study, we aim to harness the full potential of our novel monitoring system (MOTUS (MOnitoring of Transcranial UltraSound)) to improve both safety and efficacy of FUS that can be used to both target and image responses to both central (C-MOTUS) and peripheral (C-MOTUS) neuromodulation with FUS in rodents and primates. The novel ultrasound-based methodologies proposed herein would constitute breakthroughs in FUS modulation monitoring and allow for the first time accurately targeting (on the order of a few millimeters) and steering across both shallow and deep-seated regions (on the order of several centimeters in depth) as well as monitoring activity.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY. Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, autosomal recessive neurological disorder characterized by the progressive degeneration of the central nervous system. PKAN is the most common form of neurodegeneration associated with brain iron accumulation, a group of clinical disorders characterized by abnormal involuntary movements, alterations in muscle tone, and extrapyramidal signs. It is an invariably a fatal disease. Classical PKAN begins in early childhood and worsens gradually such that within the first five years of life dystonia of the musculature of the mouth, throat and tongue results progressive dysarthria, dysphagia and eventually loss of speech and impaired swallowing. Most individuals are unable to walk without assistance by age 10 to 15 years. Atypical PKAN, progresses more slowly, and appears later in childhood or early adolescence, usually after the age of 10 years. Multiple rare, completely penetrant variants in the PANK2 gene cause PKAN and are believed to alter the function of mitochondrial pantothenate kinase 2 reducing coenzyme A (CoA) primarily in the basal ganglia, especially in the globus pallidus. In 2022, physician colleagues in the Dominican Republic told us about multiple individuals and families with PKAN in an isolated region in Barahona Province and knew of more affected individuals scattered throughout the country. If confirmed, this would indicate that the frequency of heterozygous carriers of PANK2 causal variants was likely to be highly elevated. Currently there is no process for genetic testing available and there is limited genetic counseling in the country. Carrier testing for individuals at risk and their relatives, and prenatal testing for pregnancies, would be possible if all the pathogenic variants were identified. Our colleagues asked whether we could assist them in developing capacity for a clinical and research program for individuals putatively diagnosed with PKAN and for genetic testing and for prenatal diagnosis for couples at or before marriage, preconception or early in pregnancy with intensive programs of genetic counseling. The overarching goal of this project is to identify all PANK2 variants among individuals with PKAN in the Cabral- Barahona region, the surrounding villages, and in other parts of the country. We previously obtained blood for DNA extraction in affected individuals with clinically diagnosed PKAN and confirmed PANK2 variants by whole genome sequencing and sequenced their first-degree relatives to identify heterozygous carriers. Working with our colleagues, we intend to establish genotype-phenotype correlations, identify possible modifiers influencing the age-at-onset and disease type, establish a PCR-based molecular testing program in the capital in Santo Domingo, develop a community health worker-facilitated genetic counseling program, and establish the capacity for a sustainable testing and counseling program to manage PKAN and facilitate research.

NIH Research Projects · FY 2025 · 2025-08

Despite knowledge of obstetric, medical, and social risk factors that contribute to poor perinatal morbidity and mortality outcomes, significant variations in pregnancy outcomes persist across different patient populations. Healthcare delivery patterns and documentation practices vary across healthcare settings and patient populations. Natural language processing (NLP) is a data science approach that has emerged as a promising tool to analyze documentation patterns in clinical documentation. Documentation patterns can influence the quality of care provided to pregnant women. Previous NLP studies have shown that documentation patterns can vary by patient demographics and clinical characteristics. Correctly identifying documentation patterns in the electronic health record (EHR) is crucial, as they can influence the perception of subsequent clinicians reading the note. We propose a study that builds on previous work to examine documentation patterns in clinical notes from hospital birth admissions. The overall objectives of this study are to expand and refine our NLP system to accurately identify documentation patterns in EHR notes, investigate the associations between documentation patterns and morbidity outcomes for pregnant women and newborns, and inform discussions and interventions that lead to institutional change and improved patient outcomes. This secondary analysis will examine EHR notes for all ~35,000 pregnant women admitted to two NewYork-Presbyterian Hospitals from 2020 to 2024. We have assembled a multidisciplinary team with expertise in perinatal epidemiology, NLP and data science, obstetric medicine, linguistics, and community engagement to complete three aims. Aim 1: Expand and refine an existing NLP system to identify documentation patterns in obstetric clinical notes. Aim 2: Apply the NLP system to examine documentation patterns by patient demographic characteristics. Aim 3: Examine associations between documentation patterns and morbidity outcomes for pregnant women and newborns in our study sample, adjusting for sociodemographic, clinical, and care characteristics (e.g., pregnancy complications, length of stay). Outcomes include: administration of medications for preterm birth, low-risk cesarean birth, infection, hemorrhage, APGAR score, and intensive care admission. Study findings will inform future multilevel interventions, including: 1) clinical decision support to optimize documentation quality, 2) education and training of clinicians to improve documentation practices, and 3) realignment of institutional policies and processes to positively influence documentation quality and improve patient care quality.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Exposure to high acute doses of ionizing radiation as a consequence of a radiological or nuclear event can cause illnesses known collectively as acute radiation syndrome (ARS). No medical radiation countermeasure has been approved as yet by the FDA to specifically counteract gastrointestinal (GI) ARS. Radiomitigators efficacy studies cannot be conducted in humans for ethical reasons, thus the FDA requires these tests in well-controlled animal studies. Two concerns however arise; first, the results obtained from animal models do not always translate to humans: For instance, they may not reflect the inter-individual variability in the human response to radiation damage due to factors such as age, sex, and genetic predisposition. Second, in- vivo animal studies are laborious and expensive, and are not generally consistent with screening a large number of drugs or drug variants. Thus, there is a need for alternate pre-screening models which are a) based on normal human GI tissue and b) amenable to high-throughput pre-screening. To fill this gap, our goal is to develop and validate the use of human normal tissue patient-derived organoids (NT-PDOs) established from healthy normal tissue biopsies for pre-screening new candidate GI radiation countermeasures, and to assess the significance of age and sex, in a high-throughput multi-well format. In the context of GI ARS, NT-PDOs are an excellent extracorporeal model for the personalized screening of radiomitigators. They stably replicate the morphological characteristics and physiological functions of the original donor tissue, accurately reproduced the response of the human intestine to known toxic and nontoxic drugs, and when exposed to ionizing radiation, cognate organoids had similar survival dose responses as small and large intestinal crypts in mice. With two interrelated Aims, this proof-of-concept study aims at evaluating the feasibility of using NT-PDOs as proxy of the native organ to test the efficacy/safety of new candidate radiomitigators before advancing to more complex animal studies. To this end, efficacy of the test radiomitigator CBLB502 will be evaluated in organoids derived from the small intestine of individuals of different ages and sex. In Aim 1, effectiveness of the test radiomitigator will be assessed as organoid survival and viability dose-responses. The mode of action of CBLB502 has been well characterized in animal models; the goal is to evaluate whether the drug exerts similar functions in irradiated NT-PDOs. In Aim 2 for both sexes, the sample group identified in Aim 1 with the combination of age, radiation dose, and time of administration of the drug after exposure that shows the biggest effect on survival compared to respective sham-treated controls, will be analyzed using a RNAseq approach coupled with RT-PCR of selected genes of interest. The use of NT-PDOs to pre-screen medical countermeasures in a high-throughput multi-well format can complement the animal in-vivo based strategy for the assessment of radiation countermeasures for GI ARS.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Obesity produces a chronic pro-inflammatory state, and increases the risk for inflammation driven pancreatic diseases, most notably acute pancreatitis, and pancreatic cancer. The exocrine pancreas contains a high density of specialized macrophages and T cells that interact together, but little is understood about how obesity affects pancreas immune cells and their role in driving pancreatic inflammation. We therefore investigated how obesity affects pancreatic immune cells. Body mass index, a marker of adiposity, correlates with the density of TRMs in the exocrine pancreas. Obese pancreas also shows a higher proportion of pro-inflammatory macrophages expressing CD11c which interact with and increase T cell activation. Deeper analysis with single cell sequencing revealed that macrophages with signatures of embryonic origin have strong pro-repair functions, while those derived from bone marrow monocytes upregulate CD11c and show pro-inflammatory properties. This suggests that during obesity, monocyte- derived macrophages may contribute to inflammation, while embryonic macrophages attempt to maintain tissue homeostasis. To test this hypothesis, we propose two specific aims. In aim 1 we will investigate how the origin of pancreatic macrophages (embryonic vs. monocyte-derived) affects their function in obesity. We hypothesize that monocyte-derived macrophages, differentiating in obese pancreas, are more pro- inflammatory than embryonic macrophages or those found in lean individuals. To test this, we will isolate and compare the macrophage lineages from obese and non-obese organ donors and analyze their cytokine production and ability to activate T cells. Additionally, a mouse model will be used to track and compare these macrophage populations in lean and obese mice. This will ultimately reveal the source of pro- inflammatory macrophages during obesity. In aim 2 we will explore how monocyte-derived macrophages contribute to pancreatic inflammation in obesity. We hypothesize that these macrophages exacerbate inflammation and disrupt the normal function of embryonic macrophages. To test this hypothesis, we will perform multiplex staining and immune spatial profiling of obese and non-obese human pancreas to quantify the interactions of embryonic and monocyte derived macrophages with T cell rich inflammatory foci that appear during obesity. Furthermore, a mouse model for the selective depletion of monocyte-derived macrophages will be developed. This model will be used to study how depleting these macrophages affects immune regulation and T cell interactions within the pancreas of obese mice. This research leverages human data and mouse models for a deeper understanding of pancreatic macrophage origin, and function during obesity, and their role in pancreatic inflammation. By addressing this critical knowledge gap, the study holds promise for developing novel therapeutic strategies that target macrophages to prevent or treat obesity-associated pancreatic diseases.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Alopecia Areata (AA) is a chronic autoimmune disease of the hair follicle (HF) that results in hair loss. While the exact cause of AA is unknown, genetic predisposition and environmental triggers might play important roles in the disease. Recent studies suggest that dietary gluten, a known trigger in celiac disease (CD), may exacerbate autoimmune responses in AA, particularly in individuals with gluten sensitivity or CD. However, the precise role of gluten in the onset and progression of AA remains unknown. This project aims to fill this critical knowledge gap by investigating how dietary gluten, through its impact on the gut microbiome, influences autoimmune responses in AA. This research aligns with the mission of the NIAMS by contributing to a better understanding of immune-mediated diseases and informing the development of more effective and safer therapeutic strategies. The long-term goal of this study is to improve the management of AA by investigating how gluten consumption and the gut microbiome contribute to disease progression. The research aims to uncover the relationship between dietary gluten, gut dysbiosis, and immune system activation, potentially leading to novel, personalized interventions such as dietary modifications and microbiome-targeted therapies. The study focuses on two key objectives: Aim 1: Investigate how gluten-induced gut dysbiosis affects gut permeability and promotes immune activation, which drives AA. Aim 2: Explore how gluten metabolism by specific gut bacteria, such as Ligilactobacillus murinus (L. murinus), generates immunogenic peptides that activate CD8+ T cells, contributing to AA progression. This research will use well-established mouse models of AA to investigate gluten’s impact on gut health and immune function. The experimental approach includes: (1). Gut microbiome analysis: We will use 16S rRNA sequencing and shotgun metagenomics to profile gut microbiota changes in AA mouse models due to gluten consumption. (2). Gut permeability and immune activation: We will assess changes in gut permeability using FITC-dextran assays and examine pro-inflammatory cytokines and CD8+ T cell activation to link gut dysbiosis with immune responses in AA. (3). Bacterial gluten metabolism: We will investigate how L. murinus metabolizes gluten into immunogenic peptides, influencing AA progression. This research will address a critical gap by exploring the link between dietary gluten and autoimmune disease. If successful, it could lead to personalized dietary interventions and microbiome-based therapies, offering effective alternatives to immunosuppressive treatments and transforming the management of AA and other autoimmune conditions.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT My research program focuses on leveraging novel technologies to study the influence of maternal metabolism on fetal behavior during pregnancy, with a focus on understanding early biomarkers of fetal-to-infant neurodevelopment. The proposed project will implement innovative wearable devices to study the impact of maternal glucose on fetal development pathways through which typical and atypical physiology and metabolic exposures shape fetal neurodevelopmental trajectories. My training to date has provided me with a strong foundation of skills in developmental psychology, infant neurobehavioral assessment, fetal to infant electrophysiology, and biomarkers related to early life experiences. To effectively establish and lead my research program I require additional training on glucose metabolism during pregnancy, advanced deep learning techniques to analyze complex continuous data, and additional mentoring on working with term and preterm neonates in a research setting. By engaging in this protected training time, I will enter my independent stage of research well prepared to lead a research team studying the impact of fluctuations in maternal glucose on fetal behavior and autonomic maturation and their long-term influence on neurodevelopmental trajectories. Research Project: Exposure to gestational diabetes in utero is associated with an increased risk for infant neurodevelopmental delays and psychiatric disorders. While neurodevelopment begins in utero, we lack critical knowledge about sensitive periods and mechanisms that contribute to increased vulnerability in affected offspring. This K99/R00 project aims to fill this gap by examining how maternal glucose fluctuations, assessed via continuous glucose monitoring (CGM), impact the development of fetal behavioral states and autonomic regulation in healthy and hyperglycemic pregnancies, and how these may predict neurodevelopmental risks in infancy. During the K99 phase, we will employ deep learning techniques to analyze how gestational diabetes affects sequential and temporal patterns of fetal heart rate, measured via fetal electrocardiogram (fECG), to evaluate fetal behavior and autonomic maturation (Aim 1). Additionally, we will examine how GDM affects the development of cyclicity and stability of fetal sleep-wake cycles in continuous overnight recordings (Aim 2). The R00 project will investigate how typical and atypical fluctuations in maternal glucose throughout pregnancy shape the development of fetal behavioral states and autonomic control, with neurobehavioral evaluation extending to 6 months after birth (Aim 3). By integrating wearable technologies such as CGM and fECG for continuous, naturalistic assessments, this project offers an unprecedented opportunity to capture the impact of glucose fluctuations on fetal behavior. This approach will lay a foundation for identifying sensitive periods, underlying mechanisms, and early indicators of neurobehavioral trajectories.

NIH Research Projects · FY 2025 · 2025-08

To rigorously test the Developmental Origins of Health and Disease (DOHaD) research model, this project will leverage the spectacular scientific advancements of in vitro fertilization (IVF) and compare maternal prenatal distress effects between IVF donor oocyte/embryo and non-donor oocyte pregnancies. This will be the first study to use multidisciplinary (neurobehavioral, epigenetic, transcriptomic) methods with adoption-at-conception preg- nant individuals to determine whether prenatal programming can be detected independent of shared maternal- child genes. In a longitudinal study of 2nd trimester pregnant individuals (60 donor oocyte/embryo,120 non-donor oocyte), n=180 post attrition, protocol is: Zoom-based psychosocial questionnaires 26-28 weeks (Pregnancy Session 1); laboratory session for maternal EKG, blood pressure, blood draw (immune markers), psychosocial questionnaires, fetal neurobehavior 34-36 weeks (Pregnancy Session 2); collect placenta and cord blood (corti- sol); newborn EEG in hospital; medical record data; and, methods: placenta targeted/genome-wide methylation, gene expression, distress, Allostatic Load (AL): Test three aims, identifying, independent of IVF group sta- tus: Aim 1: the influence of maternal distress on perinatal neurobehavioral development. Hypotheses: Independent of IVF group status, higher maternal AL will be associated with (a) higher 3rd trimester FHR reactiv- ity, lower FHR variability, lower FHR-movement coupling and (b) newborn reduced high-frequency EEG power, greater frontal alpha asymmetry, and smaller P3 component of the ERP to novel auditory stimuli. Aim 2: maternal distress affecting placenta gene methylation. Hypotheses: Independent of IVF group status, maternal AL will be associated with placenta (a) differential DNA methylation in glucocorticoid-regulating genes (NRC31, FKBP5, HSD11B2), (b) epigenetic differences using a novel hypothesis-generating method that reduces testing burden and will improve our ability to detect high order regulatory processes across the genome. Aim 3: maternal experiences associated with unique placenta transcriptomic profiles. Hypotheses: Independent of IVF group status, maternal AL and well-being each will be associated with unique placenta gene expression in (a) pro-inflammatory genes, (b) transcriptome-wide co-regulated modules, and (c) (exploratory) causal pathways in placental biology and perinatal development using multi-omics integration. This proposal is significant because it will move DOHaD science forward, carefully testing a key gap in current knowledge, a genetic confound, and potentially producing findings (1) relevant to policy initiatives supporting pregnant individuals for two-generation impact, (2) validating to those in the U.S. having children via IVF — yearly over 2% of total U.S. births, over 10 million born so far worldwide, and (3), meaningful to all par- ents interested in the extent to which parenting, and its influence on children, begin before birth.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons (MNs) in the brain and spinal cord. The resulting clinical presentation is heterogeneous and exists on a spectrum ranging from pure motor involvement (ALS) to pure cognitive involvement (frontotemporal dementia; FTD) referred to as the ALS/FTD spectrum. Over the past decade, increasing lines of evidence have revealed a potential role for immunity in ALS pathophysiology; however, this association in the human system remains not well described. Mislocalization of TAR DNA-binding protein of 43kDa (TDP-43) in 97% of ALS cases, is a hallmark of ALS/FTD and leads to the generation of de novo peptides/proteins in the central nervous system (CNS). We posit that these de novo proteins, in addition to post-translationally modified ALS-specific aggregates, may be immunogenic and contribute to underlying disease pathophysiology. Furthermore, aberrant clonal T cell responses have been implicated in ALS pathogenesis, and ALS microglia are pro-inflammatory and have toxic effects on MNs. Here, we will leverage an unbiased approach to identify self- and non-self peptides/antigens presented to brain-infiltrated T cell responses in ALS/FTD. Our overall goal is to decode the immune landscape in the brain of sporadic and familial ALS patients and control subjects through the identification of the immunogenic proteins presented by major histocompatibility complex (MHC) class I molecule expressed on microglia, mapping of their recognition by cognate T cell receptors (TCRs) in ALS/FTD. While prior efforts in other diseases (e.g., type 1 diabetes) have spanned decades to link antigens to TCRs, this proposed studies in ALS/FTD will be catalyzed by the multi-disciplinary expertise of our interdisciplinary team. We have access to postmortem tissue from ALS patients and control subjects (n=30) as well as blood cells from living ALS patients and healthy controls (n=40). We will use these specimens to identify MHC-bound peptides/antigens in the ALS/FTD brain to characterize the phenotype and clonality of brain-recruited T cells in the blood of ALS patients. To decode the immune landscape in ALS, we will address the following questions: (i) which antigens are priming T cells in the ALS/FTD brain, (ii) what are the ALS-relevant antigen-TCR pairs, (iii) what is the phenotype of clonally-expanded T cells, and (iv) how can these ALS/FTD-specific antigen-TCR pairs inform our understanding of the disease pathogenesis and progression. Leveraging the immune system to halt and/or reduce the progression of ALS/FTD holds great promise. Our lack of precision in knowing which antigens and which TCRs are relevant for ALS/FTD is a key barrier to advancing precision immunotherapies, alongside those targeting cell-autonomous mechanisms of neurodegeneration.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Synthetic biology is driving a new era of medicine through the genetic programming of living cells. One particular focus has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo. Since colorectal cancer (CRC) initiation and progression are significantly influenced by interactions between intestinal microbes and the mucosal immune system, these interactions can be specifically modulated with engineered probiotics for immoprevention and interception. Our laboratories have demonstrated that a probiotic bacteria, E. coli Nissle 1917 (EcN), when orally administered, selectively colonizes colorectal polyps and adenomas versus normal tissue in murine models, and colonizes tumors in CRC patients. We additionally demonstrated that engineered EcN can produce diagnostic or therapeutic moleclues in a model of early stage CRC. This UG3/UH3 proposal aims to expand upon this approach by engineering EcN to identify precancerous lesions, characterize novel stage-specific immune targets, and rationally design immunomodulatory strains for CRC prevention and interception. In the UG3 Phase, we will employ a tamoxifen-inducible model of biallelic adenomatous polyposis coli (APC) gene deletion (ApcFl/FlCdx2-CreERT2) to characterize the dynamics of CRC stage-specific EcN colonization and immune cell infiltration/activation. We will construct a library of violacein-producing EcN to identify multiple stages of CRC, from precancerous adenoma to invasive carcinoma. We will then determine the kinetics of adenoma progression following biallelic deletion of Apc and assess the ability of EcN to colonize precancerous and cancerous lesions. Additionally, we will characterize the immune cell repertoire of EcN-colonized precancerous lesions of varying stages using spatial transcriptomics. In the UH3 Phase, we will design and engineer immunotherapeutic bacteria for CRC prevention and interception. We will create EcN strains expressing inflammatory payloads (e.g., GM-CSF and IFNg) and deliver them to mice prior to Apc deletion to determine if EcN-based immunotherapy can prevent the development of neoplastic lesions. Furthermore, to intercept CRC progression, we will design EcN strains expressing immunomodulatory payloads (e.g., GM-CSF and anti-CTLA-4 and anti-PD-L1 blocking nanobodies) and deliver them to mice after verifying the presence of precancerous polyps. Finally, we will examine the durability of the immune response elicited by engineered bacteria by challenging mice with organoids reflective of advancing stages of carcinogenesis. The overall goal of this proposal is to develop effective probiotic-based immunotherapies that prevent and halt CRC progression by harnessing the power of engineered EcN strains. These novel approaches have the potential to significantly improve CRC outcomes and provide new avenues for cancer prevention and interception.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The highly dynamic nature of RNA structure is key to diversifying RNA function. Notably, certain RNA structures can act as immunostimulatory molecules. Our immune system uses various pattern recognition receptors (PRRs) to detect immunostimulatory RNA molecules and trigger inflammation. PRRs are present in all immune and non- immune cells ensuring a rapid and broad immune response. Many PRRs are specialized in detecting long double-stranded RNA (dsRNA) structures, classically thought to be present in the viral genome. Interestingly, our studies, as well as the studies of others, have demonstrated that aberrant sensing of endogenous (self) dsRNAs by PRRs can cause severe autoinflammatory diseases. We recently uncovered that neurons are intrinsically enriched for long dsRNAs. In homeostasis, neuronal dsRNAs stimulated PRRs to produce low (‘tonic’) levels of type I interferon (IFN), which protected neurons from viral infection. However, when dsRNA levels were dysregulated (‘too high’), dsRNAs caused pathological inflammation. Therefore, neuronal dsRNA levels must be tightly regulated within a ‘Goldilocks zone’ to prevent neuroinflammation. Furthermore, our findings suggest that neuronal dsRNAs could be an attractive therapeutic target to control inflammation. However, the identities of neuronal dsRNAs remain enigmatic. We recently identified three genes that induce dsRNAs in neurons. We demonstrated that neuron-enriched genes ELAVL2, ELAVL3, and ELAVL4 (HuB, HuC, and HuD) can increase (i) the length of 3’ untranslated regions (UTRs), (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs (e.g., MDA5, PKR, and TLR3). This finding indicates that dsRNA levels correlate with 3’UTR length, giving rise to the idea that 3’UTRs could be major sites for dsRNA formation in neurons. Indeed, neurons are well known to express the longest average length 3’UTR in the human body. The central hypothesis to be tested in this application is that long 3′UTRs serve as major sources for self-dsRNAs that activate PRRs in neurons. First, we will identify additional ‘genes’ that can lengthen 3’UTRs and give rise to dsRNAs (Aim 1), then we will determine the ‘mechanism’ of how Hu proteins lengthen 3’UTRs and increase dsRNAs (Aim 2). Lastly, we will determine the ‘functional significance’ of 3’UTRs in a disease model for Aicardi–Goutières syndrome (AGS) (Aim 3). AGS is a severe neuroinflammatory disorder that can be caused by aberrant sensing of self-dsRNAs. With the proposed aims, we will test an innovative idea that globally modulating 3’UTR length can fine-tune innate immune responses in cells. Since 3’UTRs and dsRNAs are extremely divergent across species (even between mice and humans), we will utilize various human cells (including human stem cell derived neurons), and apply cutting edge genome engineering, dsRNA imaging, biochemistry, and novel dsRNA sequencing technologies our lab developed. This is a highly collaborative project at the intersection of immunology, RNA biology, and neurobiology. We envision that these studies can lead to novel therapeutic strategies that target RNA or RNA- associated pathways to control inflammation in neural and autoimmune disorders.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This proposal is in response to NOSI NOT-AI-23-001, which calls for new point-of-care HCV diagnostics. Most of global HCV burden is concentrated in low- and middle-income countries (LMICs), but the U.S. has witnessed a steady increase in infection rates in the past decade, with an estimated 140,000 new cases annually. Whereas direct-acting antivirals (DAAs) can over 95% of HCV-infected individuals, and are becoming more accessible, about 40% of infected people are unaware of their status. The current two-step diagnostic process requires an initial antibody screen followed by an expensive and time-consuming RNA test to confirm active infection. This cumbersome workflow requires multiple office visits for patients, resulting in delays in treatment initiation and significant patient follow-up loss. Our lab has been developing plasmonic-based thermocycling into a clinically useful method for fast multiplexed RT-PCR at the POC. In this approach, heating is achieved not externally via the Peltier effect, but rather internally via infrared excitation of nanoparticles; as a result, the heating is rapid, and powered with low- power robust optical components. We have gathered substantial preliminary data to validate the premise of this approach to work on clinical specimens (saliva and nasal swabs); the results (published in Nature Nanotechnology, 2022) showed the ability, within 25 minutes from sample to result, to detect SARS-CoV-2 with high sensitivity and specificity. Moreover, even in the presence of the plasmonic nanoparticles, we showed that real-time qPCR could be performed with accurate quantitative determinations of cycle threshold (Ct) values. This capability for sample-to-result analysis is faster than traditional PCR approaches using Peltier heating, and the instrumentation using readily available optics renders the approach suitable for community testing sites where rapid and accurate results are sought. This proposal integrates plasmonic PCR with plasma filtration (starting with whole blood) and magnetic bead-based nucleic-acid extraction (a technique we recently published called PRECISE, Lab Chip, 2024). The proposed combined method, PRECISE plasmonic PCR, takes advantage of special nanoparticles to facilitate a seamless sample-to-result workflow for the end user, suitable for the target community health settings to facilitate HCV diagnosis in a single patient visit to enable on-the-spot treatment. The target metrics would be the best in class among HCV nucleic-acid tests, meeting all of the criteria in the target product profile recommended by the NOSI for an “optimal” test except for the 15-minute turnaround time (our target is 30 minutes), and surpassing all “minimal” targets. The target metrics include cost considerations.

NIH Research Projects · FY 2025 · 2025-08

SUMMARY A mass casualty radiation event, such as the detonation of an improvised nuclear device or radiological dispersal device, could lead to severe hemorrhage, multi-organ failure, and infection, potentially leading to sepsis and/or death. The hematopoietic system and the gastrointestinal (GI) tract are among the most vulnerable tissues to radiation injury. High-dose radiation results in GI subsyndrome characterized by the destruction of the mucosal layer, intestinal epithelial barrier dysfunction, and aberrant inflammatory responses that could lead to rapid death. Although progress has been made to counteract the immediate effects of hematopoietic acute radiation syndrome, no FDA-approved countermeasures exist that can treat radiation-induced GI injury. Development of effective medical countermeasures (MCMs) for mitigation and treatment of GI-acute radiation syndrome (ARS) requires suitable animal models. The minipig model of ARS is becoming considered as an animal model for studying the radiation-induced GI subsyndrome, as it shares many anatomical and physiological parameters with humans. Importantly, GI injury clinical symptoms after radiation exposure are similar between minipigs and humans. In addition, identification of early surrogate markers of candidate radio-mitigators that could potentially be used as secondary endpoints is of large importance. To meet the critical need for GI-specific MCMs, Synedgen Inc., has developed a glycopolymer radiomitigator (MIIST305) that is specifically targeted to the GI tract that could potentially ameliorate the deleterious effects of radiation. MIIST305 has been shown to reduce cell death, suppress local and systemic inflammation, and confer significant survival in mice and rats exposed to high-dose partial body X-irradiation, when administered 24 hours post-irradiation. The primary objectives of this proposal are to evaluate the efficacy of MIIST305 for mitigating acute GI injury (histopathology) and inhibiting an excessive inflammatory response in the Sinclair miniature swine partial body irradiation (PBI) model (Aim 1), test a protein panel of radiation-responsive GI injury biomarkers of intestinal epithelial tight junction permeability, microbial translocation, and local and systemic inflammation in blood, fecal, and intestinal tissue samples collected from Sinclair minipigs (Aim 2), and use state-of-the-art machine learning methods to identify the top GI biomarkers that correlate with GI injury that can be developed towards a protein bioassay to predict the severity of radiation-induced GI-ARS after administration of MIIST305 (Aim 3). This proposal is relevant to this Notice of Funding Opportunity, since it focuses on the development of a MCM (MIIST305) to mitigate radiation-induced GI injury in a relevant large animal model of ARS, and the identification of early surrogate blood and fecal markers that could accurately predict MIIST305 radiomitigating efficacy. Successful completion of this study combined with our previous work on rodents will help advance MIIST305 for potential MCM marketing approval via the FDA Animal Rule.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Cardiac allograft vasculopathy (CAV) is presently the leading cause of morbidity and mortality following heart transplantation. While the pathophysiology of CAV is still unclear, converging lines of evidence point to a critical role of local immune responses in the graft tissue in this complication. In human CAV, B cell and antibody- producing plasma cell (PC) infiltrates are consistently observed in or around coronary arteries, yet these infiltrating cells are still poorly characterized. In particular, the antigen specificity and effector functions of locally produced antibodies are currently unknown. Understanding how these antibodies contribute to mechanisms of CAV would undoubtedly facilitate the development of new treatments. Here, we propose to use state-of-the-art immunoglobulin gene repertoire analysis, single-cell-RNA-seq combined with paired single-cell-IgH+L sequencing (BCR-seq) to obtain a comprehensive characterization of plasma cells infiltrating cardiac allografts during CAV. The functional properties and pathogenicity of individual antibodies produced in situ will also be evaluated using both in vitro cell-based assays and in vivo experimental transplantation models after generation of recombinant monoclonal antibodies from intragraft plasma cells. Aim 1. To characterize graft-infiltrating plasma cells in human CAV. Studies in aim 1 will combine IGHV repertoire and single-cell-RNA-seq analyses to determine the clonal composition and transcriptome profile of plasma cells found directly at the graft site during CAV. These experiments will also identify predominant clones expanded in situ. Using an expression-cloning platform, we will generate recombinant monoclonal antibodies from a large number of plasma cells expanded in the graft infiltrates and identify their specificity. Aim 2. To determine the function of antibodies produced by intragraft plasma cells. We will focus here on the ability of antibodies secreted in situ to form immune complexes (IC) and activate FcγR-expressing cells in the graft. Experiments in aim 2 will use a scRNA-seq approach combined with CITE-seq to comprehensively map all immune and non-immune cells expressing FcγR in the graft and therefore capable of responding to IC. We will then investigate whether stimulation of these cells through specific FcγR leads to the engagement of pro- inflammatory and pro-fibrotic pathways associated with CAV. Lastly, we will assess whether antibodies secreted in situ amid CAV can modulate the function of biologically active metabolites they react to (e.g., bilirubin). Aim 3. To assess the capacity of antibodies produced by intragraft plasma cells to promote CAV in vivo In aim 3, we will use FcγR humanized mice and a heterotopic heart transplantation model to assess the capacity of antibodies secreted by graft-infiltrating PC to contribute to CAV in vivo. Moreover, we will use a series of constitutive or conditional knockout strains to determine which cells and FcγR are implicated in the effect. We will particularly investigate the involvement of the neonatal Fc receptor FcRn expressed by graft cells as this receptor was recently involved in IC-mediated inflammatory reactions in autoimmune diseases.