Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Hypertension is the leading modifiable cardiovascular (CVD) risk factor, affecting 1 in 4 adults worldwide, of which 2/3rd live in low- and middle-income countries. Despite the availability of effective interventions, 46% of adults with hypertension are undiagnosed; and only 1 in 5 who are aware of their diagnosis are adequately managed. South Asia has one of the highest prevalence of hypertension in the world, and only less than 10% adults with hypertension achieve blood pressure control. Patients living in urban areas at particular high risk of complications of hypertension due to a variety of reasons including complex urban health system, loose social cohesion, stress and limited access to physical activity and healthy food and increased exposure to pollutants. We propose a multimodal intervention that leverages telehealth and community health workers to connect patients with severe hypertension to primary care resources and coach them using evidence-based, practical lifestyle solutions relevant to urban living. We call the intervention Coaching and Navigation by CHW through Telehealth for High-risk Hypertension or CONNECT-HTN. We have three specific aims. We will first iteratively refine and finalize the protocol for implementing the CONNECT-HTN, including establishing community and stakeholder engagement to ensure the sustainability of this approach. We will then determine whether the use of CONNECT-HTN is effective in reducing the rate of death and hospital admissions due to heart disease or stroke and compare it to referral to clinic-based care. In parallel we will evaluate the implementation of our intervention using a convergent, mixed methods study design and the Consolidated Framework for Implementation Research (CFIR). Our expectation is that CONNECT HTN will add to the evidential basis for implementing many of the WHO Best Buys for Non- Communicable Disease (NCD) prevention and control and will be the first study powered to measure substantive mortality and mortality outcomes in LMICs

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Infantile epileptic encephalopathy (IEE) is a severe form of early-onset epilepsy and has been associated with de novo and inherited mutations in HCN1 (hyperpolarization-activated and cyclic-nucleotide gated) channels. Current drugs available to treat IEE are nonspecific. To create specific pharmaceuticals for IEE, disease-related polymorphisms have been cataloged and pathology has been attributed to aberrant trafficking to the cellular membrane or alteration of gating properties of HCN1 channels. The overall goal of this application is to develop a novel targeted approach to uncover druggable HCN1 allosteric sites using genomic, functional, and structural approaches. We propose to utilize both an allosteric inhibitor and an allosteric activator of HCN1 to uncover their mechanism of action by structural determination of complexes and investigation of their functional effects on WT and mutant channels. We will then determine the association of allosteric trajectories predicted by coevolution models with the mechanisms of action of these small molecules as well as pathogenic mutations in HCN1. Together with determining structures of other gating states, this will allow generation of a model of ion channel allostery and will provide a testable framework for structure-function relationships. Allosteric modulation of HCN channels will thus be predicted and exploited for rational design of candidate therapeutics. Our first and second specific aims are to determine the structures by single-particle cryo-EM of HCN1 in complex with an allosteric inhibitor, propofol, and in complex with an allosteric activator, PIP2, respectively. The candidate binding sites will be validated by mutagenesis followed by electrophysiology. Clues into the molecular reasons for the HCN1 selectivity for propofol despite strong sequence conservation across all HCN family members, will be provided by a bioinformatic co-evolution analysis. Statistical coupling analysis will identify and predict HCN1 allosteric pathways and we will determine their association with pathogenic missense mutations and propofol binding. Candidate positions will be tested by mutagenesis and electrophysiology. Our third specific aim is to obtain structures of distinct conformers of HCN1 channels by adjusting the sample conditions (e.g. liposomes with an established electrochemical gradient) as well as by targeting disease-associated HCN1 polymorphisms. This aim will not only aid in understanding the basics of channel gating by determining physiologically-relevant channel conformations, but also will facilitate structure-based drug design to modulate aberrant, disease-specific ion channel activity.

NIH Research Projects · FY 2025 · 2024-03

Program Summary I am an early-stage investigator and recent KL2 scholar who has used econometric and spatiotemporal meth- ods to examine the propagation of C-19 in vulnerable populations. The focus of this R03 is to build on this work to examine the interaction between two devastating public health emergencies: the COVID-19 (C-19) pan- demic and climate change-amplified extreme heat events (EHEs). Although coronaviruses in general survive longer in environments of lower humidity, temperature, and sunlight, C-19 propagation has surged in summer months. A proposed explanation is that SARS-COV2 remains stable in hotter, humid environments, and that C- 19 transmission is promoted by heat-avoidant behavior that increases indoor physical proximity and air condi- tioner use. EHEs adversely affected one in five Americans. Thus, EHEs may intensify C-19 propagation, partic- ularly among more individuals and subpopulations vulnerable to both C-19-related and EHE-related morbidity and mortality – older aged individuals with medical comorbidities, socioeconomically disadvantaged individu- als, and minorities. An examination of this interaction will provide the first evidence of the association between climate-amplified EHEs and C-19, providing important data for future pandemic preparedness and climate-am- plified infectious disease propagation – a critical area of inquiry as described by several institutions, including the Federal Government. This proposal’s central objective is to examine the relationship between EHEs and C- 19 propagation, providing data that can subsequently be translated into future tools for pandemic prepared- ness in the age of climate change. My central hypothesis is that EHEs increase C-19 risk by increasing house- bound populations and promoting SARS-COV2 transmission dynamics, particularly in areas with higher pro- portions of older aged individuals, racial/ethnic minorities, and other socioeconomically disadvantaged individu- als. I will test this hypothesis by employing multivariable spatiotemporal models with quasi-experimental de- signs on several secondary data sources from the Johns Hopkins Coronavirus Resource Center (daily, nation- wide, county-level C-19 outcomes) and patient-level data from the NCATS National COVID Cohort Collabora- tive (N3C), combined with area-level socioeconomic data from the US census, and environmental data from the National Weather Service (NWS) from 2020-2022. The aims are: 1) To examine the association between EHEs and county-wide C-19 risk from a national perspective; and 2) Identify adult individual-level demo- graphic, clinical, and area-level socioeconomic characteristics associated with increased risk of C-19 related hospitalization after EHEs. This work is highly relevant to the National Institutes of Health (NIH) Strategic Framework for Climate Change and Health Initiative. In addition to supporting my pathway to independence as a translational physician-scientist, findings from this proposal will lay the foundations of future work focused on infectious disease epidemiology and climate change in a future R01 submission to the NIEHS or NIAID. 1

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Telomeres are specialized protein-DNA structures that protect the ends of linear chromosomes and maintain genomic stability. Maintenance of adequate telomere length is thus essential for cellular immortalization and tumorigenesis. While most human cancers achieve this through telomerase activation, ~ 5% - 10% of them adopt a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to elongate their telomeres. Although the ALT-driven cancers are generally aggressive with poor prognosis, there are currently no targeted therapies. In human cancers, ALT is strongly associated with genetic alterations that affect histone H3.3 chaperone ATRX-DAXX complex. We and others previously demonstrated that the ATRX-DAXX complex is essential for normal telomere maintenance. We showed that ATRX or DAXX loss, while promoting tumorigenesis by potentiating the ALT-driven immortalization, also creates a persistent telomere replication dysfunction. We therefore hypothesize that ALT-immortalized cancers must have adopted special mechanism(s) to offset their innate telomere DNA replication defects, and thus would be selectively vulnerable to the inhibition of those compensatory pathways. By combining our unique isogenic ALT-immortalization model system and customized domain-focused CRISPR screen platform, we have uncovered a list of selective molecular vulnerabilities including histone lysine demethylase KDM2A for ALT-dependent cells. We demonstrate that KDM2A-mediated H3K36me2 demethylation is required for ALT-directed telomere maintenance. Inactivation of KDM2A impairs ALT-specific multitelomere cluster dissolution, leading to chromosome missegregation and mitotic cell death. The objectives of this proposal are to delineate the molecular mechanism underlying ALT-directed telomere maintenance and to identify mechanism-based therapeutic targets against ALT-driven human cancers. To meet those goals, we will pursue the following three specific aims. In Aim 1, we will investigate the molecular functions of KDM2A in ALT-directed telomere maintenance. In Aim 2, we will define the potential adverse effects and off-tumor toxicities of future KDM2A-targeted therapies. In Aim 3, we will establish an in vivo platform to explore the synthetic- lethal interactions of ALT-driven ATRX-mutant malignant gliomas. Completion of the proposed studies will uncover the molecular mechanisms and critical dependencies underlying ALT-directed telomere maintenance and thus drive the development of novel mechanism-based therapeutics against ALT- dependent cancers including the ATRX mutant malignant gliomas.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Prostate cancer is the second leading cause of cancer death and affects about 13 out of 100 American men. The standard diagnosis approach for prostate cancer incorporates prostate specific- antigen (PSA) blood test, digital rectal examination and biopsy, and, recently, the Prostate Imaging Reporting and Data System (PI-RADS) that localizes and stratifies the risk of lesions in biparametric MRI (bpMRI) images. Detection of clinically significant cancer (csPCa) remains a challenge, resulting in over- and underdiagnosis. Thus, accurate diagnosis and staging of prostate cancer will have a significant impact on successful treatment planning and clinical management. As large-scale imaging datasets become widely available in clinical settings, more prostate cancer studies are adopting machine learning (ML) and deep learning (DL) techniques. However, the common approach in the existing studies has a drawback of using imaging findings, e.g., lesion size and radiomics features. This project aims to implement and validate cutting-edge DL-based frameworks for the diagnosis and prognosis of prostate cancer while fully utilizing imaging data and clinical scores (e.g., initial PSA level and prostate genomic score). The proposed project will use state-of-the-art DL architectures such as U-Net, image transformer and neural additive model to develop frameworks for robust DL- based prostate and zonal anatomy segmentation (Aim 1), prediction of a csPCa probability map (Aim 2), and forecast of biochemical recurrence using interpretable multimodal DL framework (Aim 3). We expect these frameworks will offer novel techniques adapted for prostate cancer, unique and interpretable features from multimodal markers to better understand diagnosis and prognosis, and will eventually help guide treatment selection and clinical management. This project will build on Dr. Kim's quantitative background in modeling and analysis of neuroimaging data. During the project, Dr. Kim will gain clinical expertise in prostate cancer via formal training driven by coursework (e.g., oncology and biology) and research mentorship. Through the K25 award, Dr. Kim will lay the groundwork for establishing an independent research program of computational methods for prostate cancer.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Mycophenolic acid (MPA) is a crucial immunosuppressive medication that prevents kidney transplant rejection and prolongs the survival of the transplanted organ. MPA, however, is associated with dose-limiting side effects of diarrhea and leukopenia. Recent animal studies have shown that MPA-related toxicities are dependent on the gut microbiota, but our understanding of MPA reactivation in the gut and its contribution to associated toxicities is incomplete. Understanding the impact of gut-mediated MPA reactivation is critical because transplant physicians frequently encounter MPA-associated toxicities in clinical practice. Transplant physicians routinely decrease the dosage of MPA during episodes of post-transplant diarrhea and leukopenia, but large studies have shown that such actions are associated with acute organ rejection. Thus, it is imperative that we define the roles played by gut bacterial beta-glucuronidase (GUS) enzymes in MPA reactivation, enterohepatic recirculation, and associated toxicities. The overall goals of this proposal are to identify the microbial GUS enzymes involved in reactivation of MPA and to define the relationship between fecal GUS activity, MPA enterohepatic recirculation, and MPA-associated toxicities in kidney transplant recipients. We hypothesize that specific microbial GUS enzymes drive the reactivation and enterohepatic recirculation of MPA, and that quantitative fecal GUS enzyme assays will serve as a biomarker for MPA-associated toxicities. Here we will study a cohort of 210 kidney transplant recipients with and without MPA-associated toxicities. Our center at Weill Cornell routinely performs approximately 250 kidney transplant recipients per year, supporting the feasibility of recruiting this number of patients that will sufficiently power the study to successfully test our hypothesis. In Aim 1, we will define the gut microbial GUS enzymes that reactivate MPA and the drugs that may disrupt them. In Aim 2, we will define the relationship among quantitative fecal GUS activities, MPA enterohepatic recirculation, and MPA-associated toxicities. This project will reveal fundamental data about the gut microbiota's ability to reactivate MPA, influence enterohepatic recirculation, and impact therapeutic tolerance. Importantly, it will assess fecal GUS enzyme activity as a novel biomarker for MPA-associated toxicities, allowing for the development of more personalized approaches to optimize MPA efficacy and minimize MPA-associated toxicities in kidney transplant recipients.

NIH Research Projects · FY 2026 · 2024-03

Project Summary The human intestinal tract supports a complex microbial environment consisting of bacterial (or microbiota) and fungal (or mycobiota) constituents. Although the role of each of these communities has been a subject of multiple studies, the role of transkingdom interactions between fungi and bacteria in shaping host immunity and physiology has been much less explored. The chemical basis for such interactions, critical for the rational design of mechanistic studies with regards to host immunity and disease development, remain completely uncharted territory in the literature. We have established a genetic manipulation pipeline to identify gene transfer methodology and build a genetic tool for nonmodel human gut bacteria on a large scale. Via a multifactorial optimization of their conjugation/transformation conditions and targeting bacterial conserved 16s rRNA genes, this pipeline efficiently identified the gene transfer methods for multiple nonmodel gut bacterial commensals and set up CRISPR-based or gene insertion tools in multiple of them. This library of genetically targetable microbes comes from 5 different phyla. This genetic manipulation pipeline and this library of tractable commensals will facilitate our investigation of trans-kingdom microbiota-mycobiota interactions at the molecular level. A high throughput screening of bacterial metabolite libraries from gut bacteria identified metabolites with direct effect on intestinal mycobiota. We identified bacterial species and corresponding gene clusters responsible for the production of these metabolites. Our preliminary data suggest strong ties and specific molecular interactions between fungi and bacteria in the gut that have previously unappreciated role in microbial dynamics, metabolite production and immunity. We will utilize such bacterial strains and isogenic mutants in key biosynthetic pathways to target metabolites with mycobiota modulatory properties. We will use several mouse models and synthetic microbial communities to define the role of trans kingdom interaction between bacteria and fungi in modulating host immunity and colonization resistance in the gut. We hypothesize that metabolites from the human bacterial microbiota modulate the fungal communities in the gut to affect microbial composition, the microbiome function and immunity. In addition to revealing novel mechanisms of fungal-bacterial interaction at an unprecedented small molecule level, the results of this proposed investigation will illuminate potential new strategies for targeting of fungal pathogens

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract Proteasome inhibitor (PI) drugs turned untreatable multiple myeloma treatable, significantly improve the quality of life for multiple myeloma patients. There are three FDA-approved PI drugs. In additional to multiple myeloma (MM), PI drugs are also approved to treat mantle cell lymphoma (MCL), amyloid light chain (AL) amyloidosis and Waldenström macroglobulinemia, the latter two are noncurable rare disorders related to plasma cells. Nevertheless, resistance inevitably occurs. All three drugs have serious toxicities. A high percentage of patients suffer from neuropathy, a debilitating adverse effect from two peptide boronate based PI drugs, bortezomib and ixazomib. Another PI drug carfilzomib also has cardiovascular and renal toxicity. Notably, none of the PI drugs showed little clinical benefits in treating patients with solid tumors in numerous clinical trials. Studies have shown that upon treatment with PIs, a transcription factor NRF1 will be activated to express more proteasomes that renders PI drugs less or non-effective. Recent studies show that when chymotryptic β5 and tryptic β2 of the proteasomes are simultaneously inhibited, NRF1, instead of activation, forms aggregated, disarming the ability of tumor cells to resist apoptotic stress induced from proteasome inhibition. Because of the striking difference between the “chymotryptic” proteolytic preference of β5 to cleave after hydrophobic residues and the “tryptic” preference of β2 to cleave after basic residues, it is challenging to develop inhibitors that can target both β2 and β5 with similar potency. Besides, such β5β2 targeting inhibitors would also have to spare the “acidic” β1 active subunit to avoid nonspecific cytotoxicity. No such inhibitors have been reported in peptide boronate and peptide epoxyketone classes. There are three known β5β2-inhibitors in other classes, all of them irreversible, carrying unpredictable toxicities. We have now designed and characterized a group of macrocyclic peptide boronates that simultaneously inhibit β5 and β2, but not β1. These compounds elicit markedly less NRF1 activation in triple negative breast cancer cells (TNBC) than BTZ. We now aim to continue our team approach to advance the structure-guided development of β5β2 inhibitors for cancer treatment by improving their specificity and pharmaceutical properties and evaluate their in vitro anticancer activity and in vivo efficacy in animal models of TNBC. In Aim 1 of this proposal, we will conduct structure-guided lead optimization to improve β5β2-inhibitors’ potency, specificity and pharmacokinetic properties. In Aim 2, we will test β5β2- inhibitors' cytotoxicity against breast cancer line cells. Aim 3 will test the efficacy of β5β2-inhibitors in in animal models of TNBC.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY The aging vasculature and associated inflammation converge as a powerful force to cause two of the most common cardiovascular conditions in older adults aged at least 75 years—heart failure with preserved ejection fraction (HFpEF) and acute myocardial infarction (AMI). Despite the negative impact of HFpEF and AMI on morbidity and mortality among older adults, there are no evidence-based strategies to prevent HFpEF and AMI among older adults. There is a strong biologic rationale that statins could prevent HFpEF and/or AMI events, given their capacity to mitigate endothelial dysfunction, suppress inflammation, and prevent ischemia. However, no randomized controlled trial (RCT) to date has examined whether statins can prevent incident HFpEF and/or AMI events in adults aged at least 75 years—the subpopulation at greatest risk for these events. The objectives of this proposal are: (1) To determine whether statins prevent incident HFpEF hospitalizations in older adults; (2) To determine whether statins prevent AMI events in older adults; (3) To determine the impact of relying on diagnosis codes to identify HFpEF and AMI. This project is an ancillary study to the ongoing NIH-funded Pragmatic Evaluation of Events And Benefits of Lipid-lowering in Older Adults (PREVENTABLE) RCT, which is examining the risks and benefits of statins in adults aged at least 75 years. Given its size (N~20,000), duration (~4 years follow-up), and study design (double-blinded, placebo-controlled, randomized trial), PREVENTABLE offers an unprecedented opportunity to examine whether statins prevent incident HFpEF and/or AMI events. However, the parent RCT’s reliance on diagnosis codes to ascertain cardiovascular events will not allow detailed insights into HFpEF or AMI given the inherent limitations of diagnosis codes to detect these events. This ancillary study will directly overcome this limitation by adding expert-adjudicated outcomes through medical record review. This study will address a huge unmet clinical need with high likelihood of informing clinical practice, and extend the impact of PREVENTABLE with minimal additional burden on study sites or participants. Our team is uniquely qualified to carry out this proposed study given our extensive experience with medical record retrieval on a national scale and with cardiovascular event adjudication; and expertise in cardiovascular epidemiology, HFpEF, AMI, geriatric cardiology, and clinical trial biostatistics. The long-term goal of this research is to identify prevention strategies for two of the most common cardiovascular events among older adults. The expected outcomes of the proposed research are 1) potential guideline-altering evidence on the effect of statins on incident HFpEF and AMI in older adults; and 2) foundational data for optimizing the use of diagnosis codes for clinical, administrative, and research purposes for studies of HF and AMI. This research directly addresses NHLBI Strategic Vision Objective 5 to “develop and optimize novel… therapeutic strategies to prevent… diseases.”

NIH Research Projects · FY 2026 · 2024-02

The Weill Cornell Graduate School (WCGS)—an educational and training partnership between Weill Cornell Medicine and Memorial Sloan Kettering Cancer Center—is a premier institution for PhD training in the biomedical sciences. We look to leverage our training capacity to develop doctoral students as leaders in the biomedical workforce. To accomplish this goal, we propose a Weill Cornell IMSD program with the following objectives: (1) Attract a pool of well-prepared students to WCGS; (2) Build a robust, self-renewing cohort of doctoral students through an early start program, interdisciplinary mentorship, and social gatherings; and (3) Equip IMSD scholars with leadership training, professional skills and networks to be leaders in a variety of biomedical and science-related careers. WCGS is primed for training the next generation of biomedical scientists, and we are confident that through the Weill Cornell IMSD, we can develop a steady stream of well-trained PhD’s that will bring innovation to the biomedical workforce.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Natural killer (NK) cells have been shown to play a dominant role in the immune-mediated control of viral infection in both humans and mice. Individuals lacking NK cells or NK cell function succumb to fatal viral infections, such as human cytomegalovirus (HCMV). Similarly neonatal mice, which lack mature peripheral NK cells, and adult mice with NK cell deficiencies are extremely susceptible to murine cytomegalovirus (MCMV) infection. Given the clinical significance of HCMV, MCMV infection in mice represents an appropriate model to study NK cell-mediated antiviral immunity. While NK cells are members of the innate immune system, it is now appreciated that NK cells share many characteristics with CD8+ T cells and can exhibit features of adaptive immunity. Although our understanding of the innate and adaptive features of NK cells has increased in the past decade, the molecular and transcriptional control of their development and optimal antiviral response remains unclear. Given the shared characteristics between NK cells and CD8+ T cells and that Wnt signaling in CD8+ T cells is instrumental for survival and in mediating responses against pathogens, I postulate that Wnt signaling plays an essential role in NK cell survival and antiviral function. However, the role of Wnt signaling in NK cells is largely unexplored. Thus, this proposal seeks to explore 1) how Wnt signaling is mediated in NK cells, 2) what Wnt ligands are modulating NK cells and what is the source of these ligands, and 3) what are the molecular events orchestrated by Wnt signaling in NK cells during development and host antiviral defense. While profiling the Frizzled (Fzd) family of receptors that bind to Wnt ligands, I found that Fzd5 is prominently and exclusively expressed on NK cells compared to other immune cells. To investigate the role of Fzd5 in NK cells, I generated a novel transgenic mouse containing NK cell-specific deletion of Fzd5. In preliminary data, Fzd5-deficient mice had diminished NK cell numbers and, strikingly, in a mixed bone marrow chimera, Fzd5-deficient NK cells were less capable of repopulating mice compared to wildtype NK cells. Additionally, Fzd5 is essential for proper antigen-specific clonal expansion of NK cells in response to MCMV infection. In Specific Aim 1 I will identify the downstream mediators of Fzd5 signaling and identify the Wnt ligand that binds to Fzd5 on NK cells. In Specific Aim 2 I will investigate the molecular mechanism of Wnt signaling in NK cell antiviral responses. At the completion of this F31 we will gain key insights into the complex transcriptional networks that are induced by Fzd5. The mechanistic insights derived from this study will be instrumental in the development of novel therapies that enhance NK cell function and influence strategies aimed at improving antiviral therapies.

NIH Research Projects · FY 2026 · 2024-02

Our lab’s research is centered around technologies involving isoform research in mouse and human tissues. Thus, we devised the first long-read approach for thousands of single cells (Single-cell isoform RNA sequencing, ScISOr-Seq, Gupta, …, Tilgner, Nat Biotechnol’18). We then devised novel statistical isoform- testing and the first spatially defined long-read sequencing approach (Slide-isoform sequencing, Sl-ISO-Seq, Joglekar, …, Tilgner, Nat Comms’21 – funded by NIGMS). We complemented this R package with a visualization tool for single-cell isoform expression (ScISOr-Wiz, Stein, …, Tilgner, Bioinformatics’22 – funded by NIGMS). Most recently, we are applying ScISOr-Seq across five adult mouse brain regions and three developmental time points to define the developmental timelines leading to brain-region specific splicing (Joglekar, …, Tilgner, to be submitted to Nature by March – in smaller parts funded by NIGMS). We devised methods to remove intronic cDNAs and artifactual non-barcoded cDNAs introduced by 10xGenomics, enabling single-cell splicing research in frozen tissues (Single-nuclei isoform RNA sequencing, SnISOr-Seq, Hardwick, …, Tilgner, Nat Biotechnol’22 – funded by NIGMS). We recently enhanced our SnISOr-Seq to define dysregulation of AS in neurons and glia from frontotemporal dementia (FTD) cases. We show that the strongest FTD-associated splicing events occur in restricted cell subtypes and that ~20% of these events detected in a cell type cannot be observed in all cells jointly (Belchikov, …, Tilgner, in review – funded by NIGMS). Separately, we defined error sources in long-read data. Specifically, we used barcoding technologies to sequence two cDNA representations of the same RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We found highly specific error patterns (Mikheenko, …, Tilgner, Genome Research’22 – funded by NIGMS). Using the knowledge on such error patterns, we then created accurate isoform detection software (Prjibelski, …, Tilgner, Nat Biotechnol’23). We have described the field in reviews on long-read sequencing (Hardwick, …, Tilgner, Frontiers Genetics’19) and single-cell isoform research (Joglekar, …, Tilgner, in review – funded by NIGMS). Upon invitation by Nature Methods, in honor of their naming of long-read sequencing as the method of the year 2022, we detailed advances and future directions (Foord, …, Tilgner, Nature Methods, in press – funded by NIGMS). Over the next 5 years, my lab will advance what is measurable in single-nuclei and spatial experiments. For example, we will define TSS at single-nucleotide, single-cell and single-molecule resolution using long reads. Moreover, we will measure DNA methylation, open chromatin, gene expression and splicing in individual cells to decipher how these layers influence each other in health and disease. More generally, we will advance what can be measured in single-cell and spatial biology, including but not limited to spliceosomal RNAs, miRNAs and protein-protein interactions that are governed by alternative splicing.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains the leading cause of death amongst infectious causes. While we have made progress reducing TB mortality, disease burden has stagnated due to our inability to reduce the incidence rate of TB. This can be attributed to our lack of transmission blocking interventions and the highly infectious nature of Mtb’ s aerosol transmission. Aerosolization represents an essential physiologic process for Mtb’ s transmission and infectious life cycle that can be targeted to reduce TB transmission and incidence. Mtb has likely evolved an adaptive response to the dramatic microenvironment changes it experiences as it transitions from the nutrient-rich lung to the desolate atmosphere. The specific genes and metabolic pathways that Mtb employs to navigate these changes are poorly understood. In the aerosol droplet environment, Mtb is deprived of CO2 and nitrogen sources which provide the carbon and nitrogen building blocks on which Mtb relies for biosynthetic processes. My project seeks to understand metabolic mechanisms underlying Mtb’ s response to CO2 and nitrogen deprivation. Preliminary data from our lab has identified arginine biosynthesis as a key metabolic pathway mediating Mtb’ s response to CO2 limitation. I propose that arginine biosynthesis mediates Mtb survival in the CO2 and nitrogen limited aerosol droplet environment, by maintaining a steady flux of carbamoyl phosphate, thermodynamically activated and short-lived essential intermediate. In this model, I propose that arginine biosynthesis is configured as a cycle mediated by ArcA or Rv2323c, a currently uncharacterized protein which I hypothesize to act as an arginine dihydrolase. Through this configuration, arginine biosynthesis couples NH4+ regeneration in ArcA or Rv2323c catalysis with the oxidative production of CO2 in the tricarboxylic acid (TCA) cycle. I hypothesize that Mtb can regulate between Rv2323c and ArcA based on nutrient availability. I predict that Mtb preferentially uses ArcA during CO2 and nitrogen limitation, since ArcA can mediate an arginine cycle without consuming CO2 and NH4+ equivalents. I will test this hypothesis with three Aims. In Aim 1, I will identify the biochemical role of Rv2323c in arginine metabolism through in vitro and in vivo characterization of Rv2323c’s catalytic activity and physiologic function. In Aim 2, I will characterize the roles of ArcA and Rv2323c in CO2 limited environments and measure their contributions to the endogenous pool of CO2 and impact on Mtb survival in air. In Aim 3, I will characterize the role of ArcA and Rv2323c in nitrogen limited environments by determining Mtb’ s preference for either enzyme in these conditions and their impact on Mtb’ s survival. These approaches will reveal novel insights into Mtb’ s adaptive response to aerosolization with the potential of identifying therapeutic targets to block Mtb transmission.

NIH Research Projects · FY 2026 · 2024-02

Abstract Cancer is among the leading causes of death worldwide and host-bacterial microbiota interactions profoundly influence tumorigenesis, cancer progression and response to therapies. Nevertheless, the role of fungi (mycobiota) in these processes remain largely unexplored, missing a potential opportunity for developing novel diagnostic, preventative, and therapeutic strategies. Across the population, colorectal cancer (CRC) is the third most common cancer type. In addition to bacteria, we recently described the presence of live, transcriptionally active fungi in colorectal cancers. Here, we preset further evidence for the presence of active mycobiota in colorectal tumors and experimental data supporting a hypothesis that fungi play an important role in colon cancer growth and response to therapies through the interaction of fungal metabolites and virulence factors in the tumor macro- and micro-environment. We will use novel methodologies and in vivo modeling allowing to determine fungal species and factors that influence CRC. We will determine specific immune mediators and cells of the immune system that interact with fungi in the tumors, and pinpoint mechanisms by which specific fungal strains influence the tumor microenvironment and impact tumor growth. We will further characterize fungi in tumors of patient and mice to determine the utility of the mycobiome as a predictor of cancer progression, survival, and response to therapies. The results of these studies will contribute towards better understanding of host-mycobiota interactions in cancer and might provide a basis for novel diagnostic, therapeutic and co-therapeutic anticancer approaches in CRC by targeting the fungal arm of the microbiome.

NIH Research Projects · FY 2026 · 2024-02

ABSTRACT Every day ~50 billion cells undergo apoptosis, or programmed cell death. Efficient recognition and clearance of these cells is critical for tissue homeostasis in physiology and for its recovery following disease. Initiation of the apoptotic cascade triggers activation of phospholipid scramblases that externalize the lipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane. Recognition of PS by dedicated receptors (PSRs) on immune cells is the first and critical step in the clearance of apoptotic cells. Immune cells with activated PSRs create an immunosuppressive environment that can be exploited by pathogens exposing PS in an immune-camouflage strategy called apoptotic mimicry. Recent studies implicated members of the XKR protein family in apoptotic scrambling. Dysfunction of XKR proteins results in an inflammatory environment and autoimmune disorders while their uncontrolled activation favors oncogenesis and facilitate viral entry. Thus, it is critical to elucidate the molecular mechanisms of XKR function and regulation to understand their physiological roles and their association with pathology. Currently, only structures of non-functional XKR proteins are available, hindering our understanding of how these protein mediate PS externalization. In preliminary experiments we show that two XKR homologues, CED-8 from C. elegans and human XKR4, scramble lipids. Using cryogenic electron microscopy, we determined the 3.55 Å resolution structure of hXKR4 in a novel, likely active conformation, providing insights into a potential mechanism for lipid transport. In our 1st aim, we propose to determine the structures of hXKR4 and CED-8 in different conformations and functional states to determine the molecular bases of XKR activation. We will use our newly developed biochemical assays to probe and elucidate the functional implications of these structures. The physiological implications of these mechanisms will be tested in cell-based measurements. Our 2nd aim is to determine how XKR proteins scramble their surrounding membrane lipids. Using cryoEM we will directly visualize the hXKR4 and CED-8 scramblases in the context of a membrane to determine how they interact with and remodel the surrounding bilayer to enable lipid scrambling. Furthermore, we will investigate how changes in the physicochemical properties of the membrane support different functional states of these proteins. Our 3rd aim is to elucidate the regulatory mechanisms of XKR activity. Using in vitro and cell-based functional assays, we will determine whether and how hXKR4 and CED-8 are directly regulated by cellular factors, such as processing by apoptotic caspases, phosphorylation, or interactions with of a peptide derived from the nuclear DNA repair protein XRCC4. Collectively, our studies will lay the foundation for future investigations of the molecular underpinnings of the XKR scramblases in vivo.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY/ABSTRACT This proposal is for a five-year research career development program, focused on understanding how “neutropenic inflammation” activates the endothelium toward programmed cell death and the protective role of necrotic cell death protein receptor interacting protein kinase-3 (RIPK3) in this inflammatory environment. The candidate has been appointed Assistant Professor in the Department of Medicine at Weill Cornell Medicine. This proposal is a natural extension of the candidate's previous research in high vascular injury ARDS populations (Price DR, AJRCCM, 2021; ICM, 2021) and vascular cell death work in ARDS (Price DR, Am J Patho, 2023). It outlines a plan for the candidate to achieve his goal of becoming an expert in vascular biology and acute lung injury, extending the training of the candidate as identified here and reflected in the mentorship of Dr. Augustine Choi and Dr. Shahin Rafii: 1. Define the role of neutropenic inflammation in mouse lung injury and human organoid models, and 2. Determine a functional role for RIPK3 in inflammatory acute lung injury, and 3. Define important necrotic cell death and vascular injury biomarkers in inflammatory ARDS. The proposed experiments and the exciting training plan will impart the candidate with a unique combination of skills that will position him to transition into a successful independent physician scientist studying vascular injury in ARDS. The acute respiratory distress syndrome (ARDS) is a common, often fatal, inflammatory lung injury for which there are limited therapeutic options. The role of vascular injury as a disease-modifying factor in ARDS pathogenesis has been well established. In contrast, the functional significance of an aberrantly injured vascular cell upregulating necrotic cell death proteins, including receptor interacting protein kinase 3 (RIPK3), remains largely unexplored in ARDS investigations, representing a potential target for future ARDS therapeutics. In this proposal, I leverage my recent work highlighting neutropenic ARDS as a high endothelial stress population to propose studies that test how excessive “neutropenic inflammation” activates the endothelium toward programmed cell death and the protective role of endothelial RIPK3 in this inflammatory environment. To do this, I have developed a neutropenic lung injury model that activates the lung endothelium and promotes RIPK3 expression. In Aim1, we will define the role of neutropenic inflammation in mouse lung injury and human organoid models. In Aim 2, we will determine a functional role for endothelial RIPK3 in acute lung injury using gene targeted mice, specifically the endothelial cell-specific deletion of RIPK3 mice. And in Aim 3, we will characterize dysregulated cell death and vascular permeability pathways in inflammatory ARDS by correlate circulating necroptosis and vascular permeability proteins with mortality and clinical indices of acute lung injury. Collectively, these studies will provide novel insight into pathways of neutropenic inflammation and endothelial RIPK3 in regulating vascular injury in ARDS.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMARY/ABSTRACT Eradicating HIV from infected individuals is obstructed by the formation of a reservoir of persistent infected cells; eliminating this reservoir could allow a cure for HIV infection. Recent research provides clues to approaches for reservoir eradication. Rare, infected individuals, termed elite controllers (EC), control HIV without treatment with antiretroviral medications. Analysis of HIV reservoirs in these individuals supports a model in which most efficiently expressed HIV proviruses have been eliminated. Thus, integration sites of proviruses from ECs are found enriched in genomic regions associated with heterochromatin and transcriptional repression. This overall pattern is, however, violated by some clonally expanded infected cells with genic integrations that show ongoing viral transcription. It has been suggested that these clones may have pro-survival and/or immune resistance characteristics, but this remains poorly understood. Our team has shown that the above features can be recapitulated by the application of CD8+ T-cell pressure in a novel mouse model of long-term infection, termed the Participant Derived Xenograft (PDX) model. For this, T-cells are obtained from ECs and separated into fractions. Naïve T cells are removed, which eliminates graft-versus-host disease upon transplantation into mice. CD4+ memory T cells are transplanted into immunodeficient mice, then HIV is introduced. Autologous CD8+ T- cells are introduced or not in controls, allowing experimental assessment of CD8+ T-cell pressure. In preliminary data, in the presence of CD8+ T-cells, the selected proviral population is smaller and viral loads lower. Advanced sequencing of integration site distributions shows larger clone sizes in the presence of CD8+ T-cell pressure, and multiple features paralleling results in ECs, such as favored integration outside transcription units, integration in reverse orientation relative to host transcription, and favored integration in more heterochromatic nuclear compartments to name a few. Thus, we propose to use the PDX model, advanced integration site analysis, and further tools to address the following Specific Aims: Aim 1. To define in high resolution the integration site features that enable the persistence of HIV proviruses under extended in vivo selection by CD8+ T-cells and compare the effects of diverse approach to enhancing this pressure. We will test multiple interventions in the PDX model to increase CD8+ T-cell pressure, reverse latency or block its establishment, and characterize in detail the molecular correlates. Aim 2. To train and validate multivariate statistical models to quantify the degree of CD8+ T-cell selection on a given pro-viral landscape and use the models to infer selection in interventional clinical trials. Completion of this study will thus provide advanced tools for quantifying CD8+ T-cell pressure on populations of integrated HIV proviruses, rich information on the functions of latency and immune modulators in the PDX model and in human trials, and an evaluation of the novel hypothesis that HIV insertional mutagenesis can confer resistance to CD8+ T-cell-mediated killing.

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Myelodysplastic syndromes (MDS) are clonal blood disorders, whose pathogenesis is driven by a class of oncogenic somatic mutations in RNA splicing factors (in the genes SRSF2, SF3B1, U2AF1, and ZRSR2) in up to 80% of cases. Efforts to target these mutations in MDS with splicing modulatory drugs have been largely unsuccessful, and there is a great need for improved therapy. To this end, our group previously demonstrated that mis-spliced mRNAs induced by splicing modulatory drugs can serve as rich sources of immunogenic tumor antigens. Based on this proof-of-concept work, we hypothesize that mis-spliced mRNA isoforms induced by spliceosomal gene mutations can also generate meaningful tumor antigens that can be targeted by cognate T-cell receptor (TCR) therapeutics. The long-term goal of this project is to develop novel antigen-based TCR therapeutics for MDS with spliceosomal mutations. Spliceosomal mutations are also prevalent in clonal hematopoiesis (CH) and associated with an increased risk of progression to MDS and acute myeloid leukemia, and the project also aspires to develop safe and effective immunization strategies for these genomic subtypes of CH. The immediate objectives of the project are to identify mis-splicing-derived tumor antigens induced by spliceosomal mutations as well as their cognate TCRs. In Aim 1, we will predict such tumor antigens by analyzing RNA-Seq datasets of myeloid leukemia patient samples and performing immunopeptidomics. We will then validate candidate peptides in vitro for their HLA binding affinity and ability to elicit CD8+ T cell responses. In Aim 2, we will identify antigen-specific CD8+ T cells in MDS patient peripheral blood and bone marrow and extract their TCR sequences using dextramer-based single-cell multiomics. We will validate the potency and specificity of the identified TCRs both in vitro and in vivo. Overall, the project will (1) corroborate our previously findings that mis-spliced mRNAs are viable immunotherapeutic targets and (2) inspire novel immunotherapies such as TCR therapeutics for MDS patients and vaccines for subjects with CH. Dr. Omar Abdel-Wahab, a clinician-scientist with expertise in spliceosomal mutations in MDS and passion for mentoring future clinician-scientists, will sponsor the project. Dr. David Scheinberg, also a clinician-scientist with expertise in TCR therapeutics as well as a track-record of training multiple MD-PhD students, will co-sponsor the project. The research efforts will take place at the Sloan Kettering Institute, where its researchers have made tremendous contributions to the understanding and clinical implementation of immunotherapies into cancer medicine. Direct mentorship from Dr. Abdel-Wahab and Dr. Scheinberg, combined with the guidance provided by the Tri-Institutional MD-PhD Program, will ensure the successful completion of the proposed project and prepare the applicant for the future career path as an independent investigator.

NIH Research Projects · FY 2025 · 2024-01

Project Summary/Abstract The gastrointestinal tract is colonized by trillions of normally beneficial microbes (termed the microbiota), as well as serves as an entry site for pathogens. Therefore, the intestinal immune system must remain tolerant to foreign non-harmful stimuli meanwhile provide protection against infections. Accumulating evidence indicates that a dysregulated immune response in the intestine is causally associated with infectious, inflammatory, and metabolic diseases, such as inflammatory bowel disease (IBD). Despite these advances, the cellular and molecular mechanisms controlling protective versus inflammatory properties of immune responses within the intestine remain incompletely defined. The long-term goal of the candidate is to investigate how diverse immune and stromal cells are regulated to promote health and homeostasis in the mammalian intestine. The candidate’s recent work identified a novel transcription factor that is expressed by group 3 innate lymphoid cells (ILC3s) and specifically restrains the proinflammatory properties of ILC3s to promote intestinal health and protect against inflammation (Zhou et al., Nature, 2022). Based on this and new preliminary data, the candidate hypothesizes that this transcriptional regulator is an essential immune modulator limiting tissue inflammation and promoting intestinal health by acting across distinct cell types. The objectives of this proposal are to understand the regulation and functions of this transcription factor in intestinal health and inflammation across distinct cell types. Aim 1 will identify the direct targets of this transcription factor and how it regulates intestinal immunity. Aim 2 will investigate its functional significance in inflammation and dissect the molecular mechanisms by which it controls intestinal inflammation. Aim 3 will determine the upstream signals that modulate expression of this transcription factor, assess the impacts of intestinal inflammation on gene expression, and interrogate the physiological consequences of these context-dependent expression alterations in intestinal health and inflammation. The results from this proposal will advance our understanding of essential pathways that shape mucosal immunity and impact the pathogenesis of IBD, which could provoke the development of novel therapeutic strategies targeting this transcriptional regulator or downstream mediators in inflammatory disorders of the gut. Career goals of the candidate are to become an independent investigator at an academic institute studying the cellular and molecular mechanisms that promote intestinal health. To fulfil these scientific and career goals, the candidate has support from a multidisciplinary advisory team with extensive experience on areas related to the proposed research and mentoring trainees to independence. The candidate has developed a plan to acquire necessary technique skills, advance scientific writing and communication skills, and enhance laboratory management and mentoring abilities during the K99 phase. This K99/R00 Award will permit an outstanding training opportunity, allow the candidate to successfully complete the proposed novel research, and flourish as an independent scientist focusing on mucosal immunity and gastrointestinal health.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT The major goal of our research program is to unravel mechanisms and detect errors during processing of repetitive DNA or RNA sequences that could lead to genomic instability and cell dysfunction. A considerable fraction of the genome in nearly all organisms consists of repetitive DNA sequences. It has been suggested that these repetitive sequences play a role in several cellular processes. However, repetitive DNA sequences can adopt non-conventional DNA and RNA structures, which can be a challenge for the DNA replication, transcription and translation machinery to travel through and process correctly. Prolonged stalling of the replication forks in human cells can have severe consequences, such as genome instability that include chromosome fragility, genomic mutations and expansions of these repeat sequences. Also, repetitive sequences located in the mRNA can interfere with translation and lead to aberrant protein expression. Several neurological and muscular diseases are caused by repeat expansions. To avoid genomic instability at repetitive sequences human cells employ several mechanisms to prevent genomic alterations, cell death and diseases. BRCA1 is one of such proteins, which is involved in several molecular pathways including DNA repair and DNA replication. However, it is not clear how much BRCA1 is needed to fulfill all BRCA1 functions properly, and which back-up mechanisms are activated in cells with 50% of BRCA1 expression. In addition, repetitive sequences in the mRNA can trigger repeat-associated non-UTG (RAN)-initiated translation leading to the generation of abnormal polypeptides that form inclusions in human cells and cause several neurological, ovarian and muscular disorders. However, the mechanisms leading to cell dysfunction, cell death and repeat expansions induced by these abnormal inclusions are not clearly understood. In addition, it is not known which trans factors and mechanisms promote repeat expansion in human cells. Aberrant inclusions could sequester or dysregulate nuclear repair proteins causing DNA damage, cell dysfunction and apoptosis. In this MIRA application we seek to determine these mechanisms and DNA repair pathways leading to genomic instability in proliferating cells and large repeat expansion in non-proliferating human oocytes. The expected outcome of this studies will be new knowledge how repetitive DNA and RNA sequences induce genomic instability and cell dysfunction in so far rare studied human cells.

NIH Research Projects · FY 2026 · 2024-01

Abstract. Genome-wide association studies have identified numerous genetic variants associated with type 1 and type 2 diabetes mellitus (T1DM and T2DM), many of which might affect genes that are involved in pancreatic β cell function and survival. However, the exact biological functions and the mechanistic nature of these genetic variants remain unclear. Human embryonic stem cells (hESCs), provide, in theory, unlimited resources to generate differentiated cells to study the role of genetic factors in human diseases. Of the diabetes-associated genes identified so far, GLIS3 is the only one (other than insulin) associated with both T1DM and T2DM, and neonatal diabetes mellitus (NDM). Recently, we reported an optimized strategy to efficiently derive GLIS3+ pancreatic β-like cells. Using this platform, we found that loss of GLIS3 causes impaired differentiation toward β cells and increases β cell death. Here, we propose to test the hypothesis that genetic and environmental factors caused loss or reduction of GLIS3 impairs human pancreatic β cell generation, survival, and proliferation in both healthy and disease conditions. In preliminary studies, we have created 2 isogenic T2DM-SNP-hESCs, 2 isogenic NDM-M-hESCs, and 2 isogenic KO (knockout)-hESC lines. In addition, we identified a TGFβ inhibitor that rescues the increased death rate in GLIS3-/- β-like cells. In this proposal, we will systematically study the differentiation of isogenic T2DM-SNP-hESCs, NDM-M-hESCs, and GLIS3-KO-hESCs, exploring the generation, function, and survival of the endocrine cells in the disease conditions. Additionally, we will explore the downstream mechanism of GLIS3. Finally, we will develop approaches to improve β cell survival in T2DM conditions by targeting GLIS3 and its downstream pathways. Toward these goals, the following aims are proposed: Aim 1. Evaluate the impact of GLIS3-associated genetic variants in the generation and survival of human pancreatic β cells both in vitro and in vivo. Aim 2. Decode the molecular mechanism of GLIS3 controlling human pancreatic β cell survival. Aim 3. Rescue T2DM islets survival by targeting GLIS3 and its downstream mechanisms. This proposal will systematically analyze the biological function of GLIS3 and associated variants in human β cell's generation, survival, and proliferation. It will significantly enhance our knowledge of β cell biology, which will pave the road for developing novel therapies for T2DM patients.

NIH Research Projects · FY 2026 · 2024-01

Glutamate is the primary excitatory neurotransmitter in the human brain, responsible for cognition, memory formation, learning, and pain signaling, among other functions. Two families of integral membrane glutamate transporters are essential players in glutamatergic neurotransmission. The VGLUT family loads glutamate into the synaptic vesicles, and the EAAT family removes the neurotransmitter from the synaptic cleft following neurotransmission. The dysregulation of these transporters under pathologic conditions and neurologic disorders disrupts glutamate homeostasis leading to aberrant neurotransmission, glutamate excitotoxicity, and neuronal death. Ions play crucial roles in the function of these transporters. Electrochemical gradients of ions power the concentrative glutamate uptake and regulate transporters. In addition, transporters can conduct ions in a manner uncoupled from the neurotransmitter uptake, modulating electrochemical trans-membrane gradients. This grant proposal aims to understand the mechanism and evolution of ion-coupling mechanisms, mechanisms of the uncoupled ion permeation in health and disease, and ion-mediated regulation. We will combine bioinformatics, single-particle cryo-electron microscopy, single-molecule fluorescence microscopy, and other biophysical and biochemical approaches to pinpoint the residues, the conformational states, and the dynamic properties of transporters underlying their interactions with ions.

NIH Research Projects · FY 2026 · 2024-01

Project Summary Ischemia-reperfusion (IR) injury after perinatal asphyxia causes neurological disability, morbidity, and even death in infants. To develop therapeutic strategies, we need a better mechanistic understanding of the metabolic changes in cerebral tissue during IR and IR-driven oxidative stress. IR-associated disruptions in glycolysis, the TCA cycle, amino acid and nucleotide metabolism, oxidative phosphorylation, and reactive oxygen species (ROS) balance lead to oxidative stress and contribute to cerebral tissue injury. The initial acute injury occurs via ROS elevation whose origin is not yet completely known. Recently, we found that oxygen deprivation in the brain results in so-called reverse electron transfer in mitochondrial Complex I leading to high ROS production and dissociation of its natural cofactor, flavin (FMN). The oxygen deprivation also degrades amino acids and purine nucleotides, which causes accumulation of ammonia. Ammonia increases ROS generation by dihydrolipoamide dehydrogenase (DLD), a component of ketoacids dehydrogenases of Krebs cycle. This contributes to a cell-type specific increase of ROS during reoxygenation and redox imbalance. Our preliminary results prompt further study of this metabolic pathway in the brain IR. Our overarching objective is to determine the main contributors to redox balance of ROS during brain IR. We hypothesize that ROS imbalance in brain IR is tissue-specific and involves at least two metabolic mechanisms: 1) a ROS decrease due to mitochondrial FMN release, and 2) a ROS increase due to ammonia-dependent activation of TCA cycle DLD. We will use advanced mass spectrometry imaging to determine the spatial distribution of small molecules in brain sections obtained after IR for simultaneous visualization of various metabolites (Aim 1). In Aim 2 we will establish the role of succinate and glycerol 3-phosphate-induced RET as modulators of ROS production in brain using transgenic mouse model expressing alternative oxidase. Mitochondria from these mice do not catalyze RET and do not produce ROS at the CxI level. In Aim 3 we will evaluate the relative contribution of neurons and astrocytes to ammonia accumulation and ROS balance. We will use freshly isolated, primary cultures of neurons and astrocytes to analyze their ammonia-generating capabilities in response to lack of oxygen These new, unrecognized, and unexplored mechanisms for the regulation of ROS production in IR explain published observations showing the transient peak of ROS during brain IR. Knowledge of the molecular details and regulation of mitochondrial ROS production is vital to clarify the fundamental principles behind brain IR injury.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Background: Mammalian models of cancer have been instrumental in the development of many targeted therapies. It is, however, difficult to establish these models, such as patient-derived cell cultures (PDCC) and xenografts (PDX). As a result, most cancer types have only a limited number of models available, which do not accurately reflect the diversity of human cancer in real life. Innovation: The majority of the patient-derived models were developed using untreated primary samples with plenty of tumor tissue. The next challenge in tumor modeling will require much more difficult samples, such as minute residual tumor foci in a patient with a partial chemotherapy response. Even though cancer is a disease of unchecked cell growth in the body, normal cells paradoxically proliferate faster than malignant cells in cell culture. As a result, in the majority of PDCC models, the cancer cells eventually disappear. To address the issue of normal cell overgrowth, we are creating a tumor-specific medium (TSM) that suppresses normal cell proliferation. Preliminary data: As a proof-of-concept, we published twenty-five new ovarian cancer cell (OvCa) lines that retained the molecular, histologic, and outcome features of the patient tumors. Objectives: Our goal is to create innovative and simple culture methods that enable the creation of patient- derived cell cultures and PDX models and develop best practices for validating these models. Specific Aims: We are working on five major solid tumor types (lung, breast, prostate, kidney, and ovary) and a liquid tumor (leukemia) to illustrate that our system can be adapted to culture the full spectrum of tumor types. Aim 1: Patient-Derived Culture of Solid Tumors: We will establish patient-derived lung, breast, prostate, kidney, and ovary adenocarcinoma cultures and compare their molecular profiles with the original patient tumor. Aim 2) Patient-Derived Culture of Solid Tumors: We will establish patient-derived AML cultures and compare their molecular and phenotypic profiles with the original patient tumor. Research Strategy: We will compare the genome, transcriptome, and proteome of each cell line with the original patient tumor, existing cell lines, and tumor datasets. Innovation: By suppressing the expansion of normal stromal cells and normal epithelium, the TSM culture system can maintain cancer cell lines long-term without feeder layers, drugs, or extracts. Impact: Predicting drug activity in the clinic has always been difficult using traditional cultural models. Therefore, having access to biologically relevant lung, breast, prostate, kidney, ovary, and AML cell lines could revolutionize cancer drug development.

NIH Research Projects · FY 2026 · 2024-01

ABSTRACT Anion channels, active transporters, and lipid scramblases are central players in human physiology. These integral membrane proteins play key roles in physiology where they control a panoply of processes, ranging from epithelial salt reabsorption and neuromuscular excitability to blood coagulation, membrane fusion and repair. My long-term objective is to understand the molecular mechanisms of gating and regulation of two families of membrane transport proteins, the voltage-gated CLCs and the Ca2+-activated TMEM16s. An unexpectedly shared property of CLCs and TMEM16s is that they display remarkable functional diversity within conserved structural frameworks. Whereas both families were originally identified as chloride channels, subsequent work revealed that many CLCs are H+-coupled active transporters and most TMEM16s are dual-function phospholipid scramblases and non-selective ion channels. Missense mutations that cause dysfunction in members of both families cause inheritable disorders of bone, kidney, brain, and muscle. Thus, CLCs and TMEM16s are priority targets for the development of pharmacological tools to treat these disorders. However, a lack of understanding of how CLCs and TMEM16s function at the atomic level significantly hinders the development of such tools. For example, the rational design of compounds to treat these disorders is hampered by the lack of structural information on the specific conformations stabilized by the gain or loss of function mutations. The overarching goal of our proposal is to understand at the atomic level how CLCs and TMEM16s are regulated by physiological stimuli and to elucidate how disease-causing mutations alter their structure-function relationships. To this end, we will focus on the CLC-1 channel which is mutated in myotonia congenita, a rare muscle disorder, and on the TMEM16E scramblase, which is mutated in limb girdle muscular dystrophy and in gnathodiaphyseal dysplasia, a rare bone disorder. The limited understanding of the molecular underpinnings of CLC and TMEM16 function is further underscored by our inability to recognize the molecular origins of the functional divergence that exists within each of these two families. In this proposal, our team will focus on broadly important questions that pertain to both CLCs and TMEM16s, such as what are the evolutionary basis of their functional divergence? What are the molecular steps underlying their activation and regulation by physiological stimuli? How do disease-causing mutations alter these molecular steps? These projects are timely, within our research abilities, enabled by the state-of-the-art approaches, and supported by extensive supporting data. We expect that our proposed work will yield new insights into these fundamental questions and will provide a structural framework to interpret the effects of disease-causing mutations, which may lead to the development of targeted therapeutics.