Yale University

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Implantable neuromodulation devices for seizure control have emerged as an important treatment for medically refractory epilepsy, particularly for focal seizure disorders increasingly identified as distributed in networks not amenable for surgical resection. Current devices, however, are not suited for monitoring and modulating a brain network due to limitations in channel count, computational capabilities, programmability, and power budget. Our long-term goal is to develop a programmable, low power embedded device which can be used for the control of seizures through monitoring and modulating brain network activity. The overall objective of this application, which is the next step towards attaining our long-term goal, is to develop a programmable computational device to perform signal processing in real-time and monitor and modulate multiple brain locations within a tight power budget. In conjunction with this hardware solution, we will implement an open language and evaluate the hardware and software solutions through bench and animal studies. Our central hypothesis, based on our preliminary work, is that instead of the current approach of performing feature detection in an analog front-end or using a single application specific IC (ASIC) – which place hard constraints on programmability – a programmable and low power digital design can be achieved with asynchronous (clock- less) design. The rationale for the proposed research is that once the hardware and software solutions are developed, we will be able to more readily advance the use of devices for seizure control and for research on brain networks. We will achieve the objective of this program through three aims. In Aim 1 we will develop a processor called neuro Stream for real-time processing of streaming multichannel data. Neuro Stream will feature a reduced instruction set computer-V (RISC-V) microcontroller unit, processing elements for specific signal processing tasks, and a vector engine. Neuro Stream will be designed using asynchronous techniques, operate within a limited power budget, and support execution of a wide range of computational methods. In Aim 2 we will implement open languages neuro Octave and neuropy. Neuro Octave will be a subset of Octave, an open-source language. Neuropy will be a subset of Python. Neuro Octave and neuropy will allow users to develop their own programs for neuro Stream using familiar, commonly used, languages. In Aim 3 we will implement monitoring and control algorithms in neuro Stream and control seizures in a model of epilepsy. This contribution will be significant because it will bring the development of epilepsy devices fully into the realm of the network theory of epilepsy allowing developments in the modulation of networks to emerge and be applied for the device-based control of seizures. The proposed research is innovative, in our opinion, because in contrast to previous advances, we will develop a programmable, powerful, all-digital solution which can be extended to incorporate future research on algorithms for seizure detection, seizure forecasting and seizure control including algorithms which use modern data science methods of machine learning and deep learning.

NIH Research Projects · FY 2026 · 2024-04

PROJECT ABSTRACT Depression affects half of those suffering from Parkinson's disease (PD), significantly reducing quality of life, increasing disability and accelerating disease progression. However, traditional antidepressants are largely ineffective in PD and there are no targeted, effective treatments for depression in Parkinson's disease (dPD). Identifying new targets and evaluating new interventions for dPD is therefore critically important. A loss of synapses in neural circuitry responsible for movement is central to the pathology of PD, secondary to a toxic build-up of alpha-synuclein. Depression often occurs before the onset of motor symptoms in PD, reflecting the presence of pathology beyond motor circuitry. dPD is therefore likely neuroanatomically distinct from PD and from major depressive disorder (MDD). We will identify the synaptic- and network-level mechanisms that are unique to dPD, which could represent important new treatment targets. Specifically, we will use PET to measure synaptic density (Aim 1), and fMRI functional connectivity to measure network function (Aim 2) across dPD, PD (no depression), MDD and HC groups, allowing for the identification of synaptic- and network-level mechanisms that are unique to dPD. The proposed study builds on our existing PET/fMRI work in PD and in MDD showing a) almost 50% lower synaptic density in the substantia nigra in PD compared to HCs and b) lower synaptic density is associated with higher severity depression, as well as network dysfunction, in MDD. Further, our pilot data supports our hypothesis that there is a distinct pattern of synaptic loss in PD depression – specifically in mood- related circuitry. We also propose to target synaptic deficits in dPD using the rapid-acting antidepressant ketamine, whose primary mechanism of action is thought to be an increase in synaptic connections (Aim 3). We have shown that in MDD individuals with a synaptic deficit, a single dose of ketamine resulted in a robust increase in synaptic (SV2A) density. This increase in synaptic density was associated with a reduction in depression severity, providing the first in vivo evidence that an increase in synaptic connections underlies ketamine's therapeutic actions in humans. We will use PET to quantify synaptic density before and 24-hours after a single subanesthetic dose of ketamine in a subset of individuals with dPD to examine the antidepressant and mechanistic effects of ketamine in PD for the first time. Taking together the strong mechanistic rationale, the limitations of traditional antidepressants in PD and the substantial untapped potential of ketamine to alleviate depression in PD, the proposed work is highly significant and timely. We firmly believe that this innovative work will drive forward discovery of critically-needed targeted, effective treatments for depression in PD.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Epilepsy is a devastating neurological disorder affecting 65 million people worldwide and more than 3 million people in the United States. Seizures impairing consciousness severely affect quality of life of people with epilepsy. However, the dysfunction in brain networks associated with these seizures is not fully understood. Previous studies explored limited set of features that can partially explain the underlying network dysfunction. Additionally, none of these studies investigated the association between ictal and interictal dysfunctional network connectivity patterns. Recently, we studied EEG characteristics of seizures impairing consciousness and developed a promising machine learning approach to predict impaired consciousness in absence epilepsy. The current proposal extends more broadly to other seizure disorders and interictal cognitive deficits. My central hypothesis is that seizures impairing consciousness are associated with both transient and chronic dysfunction in the same networks and that the characteristics of the transient dysfunction can be leveraged to develop a clinical tool to predict impaired consciousness during seizures based on scalp EEG without the need for behavioral testing. To address this hypothesis, a large EEG dataset of seizures impairing and sparing consciousness (impaired and spared seizures), as well as interictal recordings will be created. The spatiotemporal and spectral characteristics of behaviorally impaired and spared seizures will be investigated (Aim 1). A clinical tool based on conventional machine learning and deep learning methods will be developed to predict impaired consciousness based on pre-ictal, ictal, and post-ictal EEG and transfer learning will be used to enable model generalization across hospital settings (Aim 2). To characterize the relation between ictal and interictal connectivity dysfunction, we will investigate functional and effective connectivity patterns in relation to impairment during and between seizures (Aim 3). It is anticipated that spatiotemporal and spectral analyses will reveal statistically significant differences between impaired and spared seizures during pre-ictal, ictal, and post-ictal periods (Aim 1). We expect the developed clinical tool will be capable of predicting impairment in consciousness regardless of seizure type and origin and will perform efficiently across different hospital settings (Aim 2). Furthermore, the predictive models are expected to identify innovative information about evolution and stability of neural representations underlying impaired and spared seizures (Aim 2). Finally, we hypothesize that recurrent transient dysfunction in connectivity due to impaired seizures will be associated with chronic connectivity dysfunction in the same networks (Aim 3). A detailed understanding of transient and chronic network dysfunction associated with seizures may lead to development of novel biomarkers and targets for therapeutic neuromodulation. Moreover, EEG-based prediction of the behavioral impact of seizures may help guide clinical decisions about treatment including medication adjustment, surgery or neurostimulation, and lifestyle factors such as driving safety.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Obesityis a global health problem that increases the risk of type 2 diabetes, cardiovascular diseases,and cancer. As the main inducible thermogenic cell type capable of improving energy homeostasis in humans, beige adipocytes are a potential therapeutic target to combat obesity and related comorbidities. A critical barrier to the anti-obesity potential of beige fat is the limited understanding of the intercellular regulation of beige adipogenesis in obesity. Adipose tissue macrophages (ATMs) have emerged as a central regulator of adipose remodeling. Here, we have identified a microRNA cluster, miR-130b and miR-301b, as an important macrophage-derived suppressor of adipose tissue beigeing and energy metabolism. We found that miR-130b is upregulated in subcutaneous ATMs of both humans and mouse models with obesity. Our recent published study showed that mice lacking miR-130b/301b globally are protected from high fat diet (HFD)-induced obesity and glucose intolerance, concomitant with increased beigeing and decreased inflammation specifically in subcutaneous fat depot. Further studies using myeloid-specific miR-130b/301b knockout mice showed that deletion of miR-130b/301b specifically in myeloid cells resulted in nearly complete loss (~99%) of miR-130b in adipose stem/stromal cells (ASCs) and in circulation, indicating that myeloid cells are the major source of miR- 130b. In vitro studies demonstrated that macrophages (Mφs) release extracellular vesicles (EVs) containing miR- 130b that are taken up by ASCs, and miR-130b overexpression in ASCs suppresses beige differentiation likely via AMP-activated protein kinase (AMPK) and mitochondrial metabolism. However, detailed molecular mechanism whereby miR-130b/301b, produced in myeloid cells, impacts beige adipogenesis and adipose tissue inflammation in specific fat depots remain to be elucidated. We hypothesize that HFD increases Mφ-derived miR-130b/301b which suppresses beige adipogenesis via EV-mediated transfer of miR-130b/301b into specific ASCs and increases inflammation via modulation of Mφ polarization. Combining in vitro and in vivo studies using Mφ-specific miR-130b/301b knockout mice and EV administration, our goals are to delineate cell-specific actions of miR-130b/301b and explore therapeutic potential of EV-mediated miR-130b/301b inhibition in obesity. Aim1 will test the hypothesis that HFD, opposed to cold, increases Mφ-derived EV uptake of miR-130b/301b into the subtypes of adipose progenitors, impairing beige adipogenesis. Cell distribution of miR-130b/301b in WT versus Mφ-specific miR-130b/301b KO mice will be assessed using miRNA flow cytometry and miRNAScope. Single-cell RNAseq and Mφ/ASC co-culture will be performed. Aim2 will assess the roles of miR- 130b/30b in Mφ polarization and the underlying mechanisms. Aim3 will explore therapeutic potential of EV- mediated miR-130b/301b inhibition in obesity and associated disorders. Results of these studies will provide new knowledge about the cross-talk between macrophages and beige adipocyte development/function in obesity and pave the way for using miR-130b/301b and their gene networks as novel therapeutic targets and biomarkers.

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract Elastin is the major component of circumferential elastic lamellae that alternate with rings of smooth muscle cells (SMC) to form lamellar units in arteries. Loss-of-function mutations in the elastin gene ELN in humans cause supravalvular aortic stenosis (SVAS), which is characterized by aortic SMC accumulation and subsequent lumen obstruction. SVAS occurs as an isolated entity or as part of Williams-Beuren Syndrome (WBS). Defective elastic lamellae and excess SMC accumulation are also observed during physiological closure of the ductus arteriosus (DA). Failure of DA closure (i.e., patent DA [PDA]) leads to blood flow imbalance and subsequent mortality. SMC accumulation is essential for postnatal DA closure and thus promoting SMC accumulation may provide a therapeutic potential for PDA. In contrast, SMC accumulation is detrimental for patients with SVAS/WBS and some congenital heart diseases in which PDA maintains pulmonary or systematic circulation. Although regulating SMC accumulation is desired in these elastin- defective arteries, mechanistic links between defective elastic lamellae and SMC hyperproliferation in SVAS/WBS and DA are not well elucidated. To address this key question, my postdoctoral studies focus on elastin aortopathy (K99), and I will bridge these findings to DA biology during the independent phase (R00). My preliminary data demonstrate that sphingosine kinase 1 (Sphk1), an enzyme that phosphorylates sphingosine into a sphingosine-1-phosphate (S1P), is the most upregulated gene in elastin mutant mouse aortic SMCs at embryonic day 15.5. This day is when differential hyperproliferative SMCs are first observed in Eln(-/-) aorta. Reduced ELN increases levels of SPHK1 in human aortic SMCs in culture and mouse aorta. Upregulated SPHK1 is also observed in mouse and human DAs. Pharmacological inhibition of SPHK1 attenuates SMC proliferation and hypermuscularization in elastin-defective aorta and DA. Although sphingolipids play a key role in vascular development and remodeling, no prior studies have evaluated the role of the sphingolipid pathway in elastin aortopathy or DA biology. SPHK-produced S1P, a highly bioactive sphingolipid, binds and activates S1P receptors. I hypothesize that in the context of SVAS or DA, defective elastin upregulates Sphk1 levels via transcription factors (TFs), leading to increased S1P binding to S1PR1 and thus, SMC proliferation. This proposal has three specific aims: 1) elucidate role of SPHK1 in hypermuscularization of elastin aortopathy (K99); 2) determine which TFs mediate elastin deficiency-induced Sphk1 transcription (K99); and 3) delineate role of SPHK1 in DA closure (R00). These studies will use induced pluripotent stem cells and aortic tissues from SVAS/WBS patients, chromatin immunoprecipitation assay in human aortic SMCs, SMC-specific Sphk1 deletion in mice and S1PR1 signaling transgenic mice. Since SPHK inhibitors are being tested clinically for cancer, modulating SPHK1 is an intriguing therapeutic strategy that warrants intense investigation.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The formation of comedones, referred to as comedogenesis, describes the production of keratinized plug that occludes the opening of the hair follicle. Comedones are often observed in common cutaneous diseases like acne vulgaris, with up to 85% of individuals experiencing comedonal lesions in their lifetime. Comedones are also seen in rare, severe diseases such as nevus comedonicus (NC), a disease involving comedones that progress into painful, inflammatory cysts. We have shown that NC is caused by somatic mutations in the NEK9 gene. Remarkably, whole exome sequencing (WES) of a common comedonal disorder, the epidermal inclusion cyst, revealed identical mutations in NEK9, suggesting NEK9 mutations may drive comedogenesis across different manifestations. While NEK9 has been well studied in the context of mitosis and cell cycle progression, sporadic associations of NEK9 with primary cilia are also seen in the literature. The primary cilium, an immotile finger-like projection from the cell with critical functions in signal transduction, houses many of the signaling pathways related to proper development and maintenance of hair follicles. Because comedones arise from follicular structures, these mutations in NEK9 provide an unmatched opportunity to elucidate the genetic and biologic mechanisms underlying comedogenesis. To accomplish this, we have generated in vitro and in vivo models of NEK9 mutations identified in NC and EIC. Our preliminary data show NEK9 mutations result in shorter cilia and less ciliation in vitro. Hedgehog signaling, a key follicular signaling pathway that acts through the primary cilium, is diminished in NEK9 mutant cells. Our immunofluorescence studies show the activated form of the NEK9 protein localizes to the base of the cilium, a new discovery in NEK9 biology. We have previously shown this activation signal to be increased across the NEK9 mutations. Together, these preliminary data support ciliary dysfunction to be a result of NEK9 mutations. To study the involvement of NEK9 and cilia in comedogenesis in vivo, we have generated a transgenic mouse using a tamoxifen-dependent, basal keratinocyte specific promoter (K14-CreERT) to drive transcription of the NEK9 mutation most commonly reported in NC. We will time transgene induction with follicular morphogenesis onset and perform immunofluorescence and advanced microscopy methods to interrogate defects in canonical follicular development. We will continue to obtain NC and EIC tissue from our institution and perform WES to identify new candidate variants that may contribute to comedogenesis. We will generate stratified skin equivalent models to continually test the newly identified mutations for ciliary phenotypes. Lastly, we will use the known biology of NEK9 to systematically test downstream effectors for possible mechanisms of ciliary dysfunction. This training proposal represents an integrated scientific approach and new learning experiences that harness techniques in cell biology, computational genetics, and pathology to yield novel insights into the mechanisms of comedogenesis.

NIH Research Projects · FY 2026 · 2024-04

Summary Intracellular environments are constantly producing local stress in the form of aging organelles or accumulations of redundant or misfolded components. During disease, these processes are exacerbated as, for example, pathogen invasion contributes another source of cytoplasmic toxicity. Our cells deal with these various challenges by creating a new organelle called the autophagosome which grows around the intracellular toxin and eventually fully encapsulates it. In this way, the dying organelle or the invading virus is sequestered from the rest of the cytoplasm. A similar process plays out when cells are starving, as autophagosomes can be used to harvest redundant material as a source of nutrients. Each autophagosome requires millions of lipids to complete its construction and in high stress, hundreds of autophagosomes may be made over tens of minutes. In just the last four years, my lab and several others in the field, have discovered the primary machinery needed to harvest most or all of the lipids involved in autophagosome construction. This machinery includes a lipid transporter and associated transmembrane proteins that distribute these lipids to both leaflets of the connecting membranes. This “bulk lipid transport” system is unprecedented and thus its discovery has raised many important next questions. Most critically, we know which machines harvest the lipids, but we do not understand how the lipids are pulled from their source membrane. We also do not have an absolute understanding of which membrane is the source. These two questions are closely connected as the ability to flux lipids out of a membrane is probably related to physico-chemical attributes of the donor membrane. In addition, the decision to flux potentially 100,000,000's of lipids out of a donor may impact the normal biology at that site and so we need to understand how autophagosome biogenesis is coupled to changes in the lipid donor membrane compartment. The other surprising element of autophagosomes is their shape. They need to adopt a bowl-like structure in order to encapsulate random bits of cytoplasm during starvation. How this occurs is not known, but several models have postulated that the harvesting of lipids alone might suffice, while others suggest key proteins that recognize extreme elements of this unique-shape will stabilize those elements driving the production of bowls. In this proposal, we build on our recent discoveries to explore where lipids are harvested from, how the flux is generated and then ask how these two activities are coupled to the formation of the bowl-like intermediates in autophagosome biogenesis.

NIH Research Projects · FY 2025 · 2024-03

Osteoarthritis (OA) is the most common joint disease and currently there is no effective means of preventing or slowing joint degeneration. Thus, identification of new OA-associated molecule(s) may provide invaluable in- formation toward the search for novel therapeutic targets for OA. Our RNAseq screen for novel, differentially expressed genes in OA led to the isolation of Nav1.7 (encoded by SCN9A) as a novel OA-associated molecule. Nav1.7 is of particular significance due to its correlation with a spectrum of hereditary human pain disorders. A nucleotide polymorphism of SCN9A was reported to be correlated with increased pain sensitivity in OA patients. Our preliminary data demonstrated that Nav1.7 was increased in superficial zone chondrocytes in both human OA cartilage and cartilage from a mouse OA model. We were excited to find that blocking Nav1.7 dramatically reduced membrane potential in human chondrocytes. To our knowledge, this is the first evidence demonstrat- ing that sodium channel has electrophysiological function in non-excitable chondrocytes. Hyperpolarization caused by Nav1.7 blockade in chondrocytes results in the alternations of secretome/cross-membrane transport of the proteins. Indeed, both direct blockade of Nav1.7 and incubation with the conditioned medium collected from Nav1.7 inhibitor-treated chondrocytes enhanced chondrocyte anabolism and inhibited IL-1β induced ca- tabolism. Further, a series of proteomics screens isolated HSP70 and midkine from the Nav1.7 blocker-treated conditioned medium as two potential key mediators of Nav1.7 in chondrocytes. More significantly, local and oral delivery of Nav1.7-specific inhibitor protected against OA and reduced OA-associated pain in both surgical- ly- and chemically-induced OA models. The hypothesis of the application is that Nav1.7 plays a pivotal role in chondrocyte metabolism and OA through regulating membrane potential and secretome profiling of chondro- cytes. The Specific Aims are: (1) To elucidate the molecular and cellular mechanisms by which Nav1.7 regu- lates chondrocyte metabolism. We will determine SA#1A) the effects of Nav1.7 blockade, overexpression and deletion on chondrocyte metabolism; SA#1B) the target genes of Nav1.7 that mediate the functions of Nav1.7 in chondrocytes; SA#1C) the molecular determinants in Nav1.7 blocker-treated conditioned medium which medi- ate Nav1.7 regulation of chondrocytes; and SA#1D) the co-factor(s) of Nav1.7 that are involved in Nav1.7 regu- lations of chondrocytes. (2) To define the importance of Nav1.7 in the initiation and progression of OA, and the underlying mechanisms involved. We will determine SA#2A) the effects of global ablation of Nav1.7 on OA ini- tiation and progression; SA#2B) the importance of chondrocyte-expressed Nav1.7 to OA; SA#2C) the depend- ence on HSP70 and midkine of Nav1.7 blockade mediated protection against OA; and SA#2D) the therapeutic effects of pharmacological Nav1.7 blockade on OA and OA-associated pain. Proposed studies will not only ad- vance the field by elucidating Nav1.7 regulation of chondrocytes and OA, but may also lead to the development of Nav1.7 inhibitors as novel diseases-modifying drugs for treating OA rather than just pain relievers.

NIH Research Projects · FY 2026 · 2024-03

Project Summary/ Abstract Nonalcoholic steatohepatitis (NASH) has reached epidemic proportions and will result in an immense burden of chronic liver disease and cirrhosis. Therapeutics for NASH is a very active area of investigation, but even with development of propriety drugs there will be major issues of financial availability for such a large percentage of the population. This motivated us to search for generic drugs with therapeutic potential in NASH, resulting in the identification of digoxin. We, and others, have identified an important role of digoxin in providing hepatoprotection in a range of liver conditions, including NASH. We have further identified that digoxin binds to pyruvate kinase M2 (PKM2) and stops PKM2 from initiating immune responses in macrophages. There is a large amount of published data from other studies that supports the ability of digoxin to reduce inflammation, steatosis, and fibrosis in the liver. There is also a high likelihood for translation of these findings to humans because we have demonstrated that oral digoxin reduces immune responses in healthy volunteers and others have demonstrated that oral digoxin reduces serum cholesterol, as we previously reported based on observations in mice. At least three mechanisms of digoxin-induced hepatoprotection have been identified. These include binding to PKM2 as reported by us, digoxin-mediated inhibition of RORγt and TH17 cells, as well as activation of transcription factor EB (TFEB). Of great clinical relevance is the fact that hepatoprotection occurs at a concentration below that required for cardiac effects, significantly improving the safety profile of digoxin. The large amount of experimental and translational data on the anti-inflammatory and hepatoprotective effects of digoxin which has accumulated over the last 55 years now supports the conduct of a clinical trial to directly test the ability of digoxin as a therapy for NASH. We will test the ability of oral digoxin to reduce steatosis, inflammation, and fibrosis in patients with NASH. This will be done at the standard cardiac dose and at a lower dose. In addition, we will examine the immunomodulatory effects of digoxin with respect to myeloid cells, TH17 cells, and liver tissue and correlate these with the clinical response. The demonstration of hepatoprotective effects of a generic medication for NASH will be transformative. Due to the lack of commercial return, this avenue of research will only occur outside the pharmaceutical industry.

NIH Research Projects · FY 2026 · 2024-03

Abstract Alcohol abuse and dependence are global health concerns associated with numerouus comorbidities. Under normal conditions glucose is the primary fuel for brain energy metabolism, but when people drink, the liver converts most of the alcohol to acetate, and the brain utilizes that acetate, partially replacing glucose consumption. Studies of hypoglycemia in diabetes and in starvation show that the transport and utilization of monocarboxylic acids are enhanced by hypoglycemia and by elevations in monocarboxlyic acids, and in single occipital volumes of the brain, we see alterations in brain acetate oxidation that are associated with drinking. Those alterations parallel certain changes that are also seen with cortisol and possibly regional brain volume loss. We now have the capacity to map brain acetate oxidation throughout the brain using a novel method called Deuterium Metabolic Imaging (DMI) together with administration of deuterated acetate, and we plan to test for relationships of regional brain acetate oxidation with cortisol, stress, alcohol-related behavior, and regional brain volumes. We hypothesize that frequent, intermittent exposure to elevated acetate can increase brain acetate oxidation globally and regionally, and that the near constant elevations of acetate in alcohol dependence has the opposite effect. We further propose that acetate oxidation is associated with brain volume deficits and cortisol levels and turnover. Our preliminary data support these hypotheses, and in this proposal we plan to test whether the condition is a state or a trait, by assessing if acetate consumption normalizes in alcohol-dependent people who have been sober for more than six months. If the hypotheses of this project are supported, the fuel-generation aspect of alcohol may provide a novel reward mechanism that promotes the continuation of binging in heavy, non-dependent drinkers and may promote prolongation of binges for caloric benefits. In dependent individuals, a reduction in the ability to oxidize acetate may exacerbate symptoms of withdrawal, which raises the possiblity of adjunct therapies of monocarboxylic acids like ketone bodies for detoxification. Because acetate is a significant contributor to cortisol synthesis, we will check for relationships between brain acetate oxidation and cortisol turnover using blood samples obtained during the DMI sessions.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY The application of machine learning (ML) to randomized clinical trials (RCTs) represents a novel avenue for developing tools to enhance precision cardiovascular care. ML-based predictive approaches can learn response profiles based on the clinical characteristics of patients included in RCTs, thereby allowing personalized inference. However, despite the development of promising algorithms from applying these novel methods to high-quality experimental data in RCTs, there is a lack of a clear pathway for their real-world evaluation and implementation. To bridge this gap, we aim to develop an implementation-aligned strategy for care personalization models. We achieve this through our three study aims. In Aim 1, we will empirically evaluate various ML approaches for RCT using adequately powered simulated heterogeneous treatment effects, specifically incorporating covariate distributions observed in real-world patients from two distinct and diverse health systems. Using participant-level data from five diverse NIH-funded RCT datasets, we will evaluate models based on their performance in detecting simulated graded positive control heterogeneous treatments effects in these RCTs as well as in “digital twins” of these RCTs, computationally designed to replicate populations of these conditions in the practice in electronic health records (EHRs). Such an approach is needed to evaluate model generalizability to different populations expected in EHRs. In Aim 2, we enhance the interoperability between RCTs and EHRs, which is required for translating RCT-derived models to EHRs as well as selecting candidate predictors based on their EHR availability. We will accomplish this by mapping covariates from RCTs to a common data model, using a novel sentence transformer to map the descriptions of these covariates to those in the common data model. We will demonstrate real-world RCT covariate distribution at 13 hospitals across two health system EHRs mapped to the same common data model. In Aim 3, we will address the informative missingness of covariates in the real-world data, representing another key challenge limiting the pragmatic evaluation of algorithms developed from RCTs. For this, we will prospectively evaluate novel approaches that adapt models for variable missingness, both random and informative, during the model development process. In this study, we will assess whether “missingness-adapted algorithms” accurately capture the personalized effect estimates for patients, compared with a complete-covariate algorithm whose covariates are captured prospectively through direct patient contact. Collectively, the proposal will develop an end-to-end strategy for evaluating models developed from RCTs to improve their selection for real-world, pragmatic evaluation and implementation in EHRs. The methods will be rigorously tested in multiple RCTs. Moreover, through open-source data sharing, the datasets and the results of our work will be available as benchmarks for the rigorous development of further methodology for detecting personalized effects from RCTs. The proposal will serve as an essential framework for evaluating and translating precision care tools developed from RCTs.

NIH Research Projects · FY 2025 · 2024-03

Project Summary: Adoptive cellular immunotherapy utilizing T cells engineered with chimeric antigen receptor (CAR) has shown durable clinical responses in hematologic malignancies. However, relapse remains a challenge, urging for better CAR designs. CAR, analogous to the T cell receptor (TCR), is specifically engineered to redirect T cell specificity towards tumor antigens. Optimal regulation on TCR dynamics is crucial for proper TCR function. Altered dynamics of TCR leads to disfunction or even immune disorders. While the importance of control TCR dynamics has been well established, there is a notable lack of comprehensive studies focusing specifically on CAR dynamics, including endocytosis, recycling/degradation, and its impact on CAR-T function. My previous work demonstrated that modulating molecular dynamics of CAR greatly improves CAR-T function. By utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CCT), I reprogrammed CAR dynamics and substantially enhanced CAR-T efficacy in vivo. To control CAR dynamics more precisely and elucidate its impact on CAR-T function, further modulation on CCT fusion and investigation on cell-intrinsic regulators to control CAR dynamics are needed. In Aim 1 of this K99/R00 application, I propose to identify the optimal position and functional motif of CCT fusion. Completion of this aim will not only develop a novel CAR engineering approach but also provide a deeper understanding of how altering CAR dynamics contributes to improved CAR-T function. In Aim 2, I plan to uncover cell-intrinsic regulator of CAR dynamics using CRISPR-based gene perturbation. This aim will serve to fill the gap in our understanding of the improved anti-tumor efficacy of CAR-CCT but also uncover novel regulators on CTLA-4 dynamics. In Aim 3, I propose to develop self-regulated CAR with CCT fusion in response to antigen stimulation. This novel engineer approach will enable precise control over the timing and duration of CAR signaling, which is crucial for maintaining CAR- T efficacy with limited toxicity. All aims center on engineering CAR dynamics with CCT fusion to develop novel immunotherapies. The innovative insights and versatile engineering approach proposed in this study will also contribute significantly to addressing the existing knowledge gap in CAR engineering. This grant will facilitate my career development during both the postdoctoral training phase and transition phase as an independent faculty and group leader.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY / ABSTRACT Despite its centrality in anxiety- and trauma-related disorders, the avoidance of danger in the environment requires new paradigms. Using the RDoC framework, the mental health field has sought to define circuit- behavior relationships that may underlie psychiatric disorders. A central challenge in this process has been the difficulty in identify clear links between threat computation and neural circuits. A further threat has been that even when such links are apparent, as in predator avoidance, the translatability of these behavioral models across species has been limited. Threat computation plays a central role in the genesis of anxiety- and trauma- related disorders, yet we do not have a clear behavioral paradigm that enables us to precisely control the perception of survival danger in the same way in mice and humans. Here, we propose to develop a novel quantitative circuit neuroscience platform for studying the fear of heights. Exposure to high places induces common patterns of peripheral arousal, avoidance, and distress across species yet has been limited in its study in mice. Since physical height is experience through the visual system, it can be precisely manipulated to induce artificial changes in threat perception that can influence behavior. Most importantly, the exposure to heights is a stimulus which induces a threat of death across species. Thus, we propose to establish a controllable model of aversion to visual heights as a new paradigm in the translational circuit neuroscience of anxiety. In order to do so, we will combine chemogenetics, fiber photometry, computational modeling of behavior, and a virtual reality system to demonstrate the role of norepinephrine circuits in the fear of heights. This comprehensive program is based on substantial preliminary data demonstrating that norepinephrine neurons in the locus coeruleus are activated by elevation. Furthermore, we will demonstrate the potential of the visual fear of heights paradigm by manipulating the perceived distance from the ground in real time, thus enabling unprecedented insights into the online computation of threat that is not possible with other fear-related paradigms. Once established, this behavioral model will facilitate inference into circuit mechanisms of innate threat that can for the first time be compared in both mice and humans.

NIH Research Projects · FY 2025 · 2024-03

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. In the oral cavity, most cases of squamous cell carcinoma begin as a precursor lesion classified by the World Health Organization (WHO) as an “oral potentially malignant disorder (OPMD).” Oral leukoplakia is the most common OPMD, with a global prevalence of 4.1% and a malignant transformation rate between 0.1%-34.0%, and the malignant transformation rate increases to 40% in oral dysplasia. These wide ranges of malignant transformation rates suggest an unmet need to develop prognostic biomarkers that can better differentiate benign from premalignant lesions and predict the risk of transformation of premalignant lesions to invasive cancer. During the development of cancer, immunoediting occurs within the tumor microenvironment. Immunoediting consists of three phases: elimination, equilibrium, and escape. We posit that there are defined immune signatures within a lesional microenvironment that correlate with the transformation of precancerous cells to invasive cancer based on the known phases of immunoediting. The current standard of care tools to establish benign from premalignant oral lesions include conventional hematoxylin/eosin (HE) and single staining immunohistochemistry (IHC), which limits the number of immune cells which can be evaluated at any one time. Thus, we developed an innovative spatial omics technology (SAFE) that facilitates the comprehensive and deep multiplexing of whole tissue sections and incorporates artificial intelligence and machine learning approaches to accelerate the analysis of ~45 molecular and immune signatures within oral lesions in a clinically appropriate timeframe. We propose to perform single-cell RNA sequencing to identify the unique cellular and immunological transcriptional programs that distinguish benign and premalignant oral lesions (N=60) and will assess relevant protein expression and spatial localization via SAFE (Aim 1). Subsequently, in Aim 2, we will evaluate the defining molecular and/or immunological signature(s) in a unique set of patients (N=55) with serial biopsies documenting transformation from premalignancy to cancer over 20 years against a separate cohort of benign and premalignant lesions (N=155). From our dataset, we will develop an oral cancer progression model that incorporates the host immune response for the first time to improve risk assessment for malignant progression to a degree superior to what is currently possible.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Human papillomavirus (HPV) is the causative agent of a growing proportion of incident cases of head and neck squamous cell carcinomas (HNSCCs). HPV type 16 is responsible for over 90% of HPV+ HNSCCs diagnosed in the United States. The HPV16 E7 antigen is an attractive immunotherapeutic target for HPV-associated cancers since it is a unique virus-specific oncoprotein constitutively expressed by all cancer cells. T cells that are genetically engineered to express an HLA-A*0201-restricted T cell receptor (TCR) that recognizes the HPV16 E711-19 epitope can mediate regression of HPV16-associated cancers in a preclinical model.1 A phase 1, first-in-human (FIH) multi-center study demonstrated the safety and efficacy of an autologous HPV TCR- engineered T cell (TCR-T) therapy which targets the HPV16 E7 binding epitope in HLA-A*0201-expressing HPV+ cancer patients (NCT03912831).2 However, the realized challenges with adoptive cell therapy (ACT) are: i) the high costs associated with manufacturability and monitoring and managing the toxicities from the supra- physiological bolus of ACT products which are combined with systemic IL-2 administration, and ii) the limited in vivo persistence due to the lack of appropriate antigen-specific stimulation due to poor intra-tumoral T cell recruitment into solid tumors. Herein, we propose innovative proof-of-concept (PoC) experiments to determine whether a low dose of adoptively transferred HPV16 E711-19 TCR-T cells can be exponentially expanded in situ by administering a novel biologic fusion protein, CUE-101, which delivers the HPV16 E7 antigen-specific stimulation with localized delivery of high doses of IL-2.3-5 Furthermore, we determine whether HPV16 E7 TCR- T cell persistence translates into an enhanced anti-tumor effect in preclinical HPV+ tumor models (Aim 1). Lastly, we build upon our team’s discovery of the role of the CXCR6:CXCL16 axis in the recruitment of peripheral circulating CD8+ T cells to perivascular niches of the tumor populated by CXCL16-expressing CCR7+ DC3s, which can sustain the survival of tumor-specific CD8+ T cells.6 Thus, we assess whether the efficacy of HPV16 E7 TCR-T cells can be improved through engineering strategies that introduce CXCR6 to the TCR-T cells, which can facilitate intra-tumoral recruitment, proliferation, and survival in situ (Aim 2). The completion of these aims will generate the supporting rationale for a future clinical trial administering HPV16 E7 TCR-T cells and CUE- 101 in HPV+ cancer patients. These efforts can potentially transform our current approaches to adoptive cell therapy.

NIH Research Projects · FY 2026 · 2024-03

SUMMARY The three-dimensional organization of the genome regulates gene expression, impacting fundamental biological processes in development, homeostasis and disease. Deep understanding of the relationship between genome structure and function requires insight into 3D chromatin architecture at individual gene loci, at high resolution and at genome-wide scale. Current approaches to visualize chromatin architecture are limited by trade-offs between depth and breadth and cannot link 3D position with sequence information with simultaneous profiling of transcription and epigenetic marks in single cells. Genomic methods such as Hi-C typically analyze a population average of genomic contact information and imaging approaches such as ChromEMT4 provide nucleosome level visualization with 1 nanometer resolution, but lack locus identification and genome scale. Thus, there is a critical need for a broadly applicable method that incorporates both sequence and nuclear position to reveal the spatial structure of the genome at nanometer scale. Ideally such a method would have nucleosome resolution (~10 nm) at the single-cell level and simultaneously provide functional readouts of epigenetic marks, transcription factors (TFs), and transcriptional activity using multimodal labeling in the same cell. The goal of this proposal is to develop, validate and benchmark such a method to provide genome-wide chromatin architecture analysis with sequence information by combining iterative expansion microscopy (PanExM) with genome-wide barcoding. This method will provide nucleosome resolution imaging of the genome with kilobase sequence resolution while allowing multimodal protein labeling of the chromatin thanks to the protein retention during PanExM. To achieve this: in Aim 1, we will adapt PanExM to visualize the chromatin (ChromExM), develop multimodal labeling of DNA, RNA, TFs and epigenetic marks, develop benchmarking tools to test isotropic expansion, structural perturbations and labeling efficiency and develop analytical and quantitative methods to characterize how local chromatin structure regulates function. In Aim 2 we will adapt fluorescence in situ hybridization (FISH)-based chromatin tracing to ChromExM using genome-wide barcodes as well as specific probes to label sites of interest such as promoters and enhancers, we will develop approaches to systematically examine enhancer-promoter interactions during transcription activation, and we will benchmark ChromExM to established methods of super resolution microscopy, chromatin tracing and HiC. The methods developed here will provide unprecedented resolution of the chromatin structure at the single cell level and genome-wide, and will be broadly applicable by the research community. ChromExM will provide much-needed tools to allow a large community of researchers to address the structural basis of how epigenetic modifications, DNA-sequence, and chromatin proteins regulate gene expression.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY: Over the past decade the development of immune checkpoint blockade (ICB) in melanoma and other tumor types has transformed the treatment options available for many patients with cancer. However, within melanoma only approximately half of patients experience a durable response to treatment, with worse results in most other tumor types. We and others have identified strategies to enhance anti-tumor immunity and sensitize non-responsive murine melanoma tumors to ICB by targeting dsRNA pattern recognition receptors (PRRs). These strategies include the agonism of the dsRNA sensor RIG-I and the loss of Adenosine Deaminase Acting on dsRNA (ADAR1). We showed that ADAR1 is an innate inhibitory checkpoint in tumor cells that limits anti-cancer immunity, and that targeting it therapeutically can trigger multiple dsRNA PRRs to sensitize tumors to immunotherapy. Both RIG-I agonism and targeting ADAR1 resulted in enhanced cytokine release in murine tumor microenvironments and improved the recruitment and control of tumors by T cells. In recent work we have uncovered evidence that co-targeting multiple dsRNA sensing pathways induces cooperative effects and enhances anti-tumor immunity compared with targeting single sensors. However, several gaps in knowledge limit the translation of these strategies into effective therapies for patients. These include: i) defining the optimal therapeutic strategy for targeting dsRNA PRRs; ii) understanding mechanisms by which dsRNA sensing in tumors programs immune cell function; and iii) identifying the patients most likely to respond to dsRNA-targeting therapeutics. To test the effects of dsRNA PRR stimulation on patient tumor and immune samples ex vivo, we developed a novel perturbational single cell RNA sequencing approach. Applying this strategy, we found that dsRNA stimulation of patient tumor and immune cells induces distinct T cell programs of activation and survival and that these approaches may be most effective in tumors with high pre-treatment levels of interferon signaling and adenosine-to-inosine dsRNA editing. In Aim 1 of this proposal, we will test the hypothesis that co-targeting ADAR1 and RIG-I enhances the activation, proliferation and function of anti-tumor T cells. We will explore mechanisms of this finding in mouse models including the increased recruitment, activation and cross-presentation of tumor antigens by dendritic cells. In Aim 2 we will seek to identify the tumor determinants of therapeutic response to targeting ADAR1 and uncover insights that will facilitate the development of small molecule and nucleic acid therapeutics. If successful, the work described in this proposal will help define the rules by which tumors and T cells respond to dsRNA stimulation and advance the development of dsRNA-targeting strategies for melanoma and other tumors.

NIH Research Projects · FY 2026 · 2024-03

Project Summary:This A1 proposal focuses on impaired pathological fracture healing in the presence of breast cancer cells, not on the entire complex sequence of cancer spread from the breast to bone. Metastatic cancer cells can settle in anywhere in the bone including bone marrow, cortical bone, or outside of the bone. Cancer cells are spilled in and out of bone at the time of fracture. Pathological fracture calluses are deficient and very often fail to ossify despite adjuvant therapies such radiation therapy and anti-resorptive agents including Rank:Fc antibody (denosumab) or bisphosphonates (zoledronate). Failed fracture healing is not solely a result of bone resorption. There is no mechanistic understanding as to why fractures do not heal well in the presence of certain types of breast cancer cells. Unraveling the mechanisms underlying impaired fracture healing will enable patients to benefit from future scientific endeavors employing mechanism-based treatments. If there is a way of addressing impaired fracture repair while also inhibiting cancer growth in bone, the clinical care for pathological fractures will be immensely impacted. We conducted whole transcriptome bulk RNAsequencing of several different types of breast cancer cells that are grown in breast vs. bone. We observed that breast cancer cells that inhibit fracture repair are pro-inflammatory with heightened MEK1-pERK1/2-cytokine- hyperinflammation signaling in the bone microenvironment. Temporal expression of pro-Inflammatory cytokines and bone-acting proteins such as TNF, interleukins, and sclerostin are completely deranged and prolonged following femur fractures in the presence of breast cancer cells such as MDA231, HC1806, and 4T1 However, there is a critical knowledge gap as to how these different breast cancer cell types affect chondro-progenitors, osteoblasts, osteoclasts, and osteocytes within the intact fracture callus architecture as suggested by the Reviewer. Two notable changes in this A1 proposal include the refinement of the animal models and a new set of compelling spatial transcriptomic biology data incorporating the Reviewers’ critiques and suggestions. We posit a central hypothesis that highly inflammatory pERK1/2-high breast cancer cells inhibit the normal fracture callus formation and maturation by causing prolonged hyper-inflammation and subsequent derangements of callus spatial transcriptomics. We propose 3 Specific Aims. Aim 1. To define temporal and spatial transcriptomic changes of inflammatory, osteogenic, and chondrogenic lineage cells in structurally intact fracture callus in vivo. Aim 2. To rescue impaired osteogenesis of osteoprogenitors, chondroprogenitors, and periosteal cells in the presence breast cancer cells by inhibition of pERK1/2-induced inflammation ex vivo. Aim 3. In Vivo Pre-clinical Therapeutic Translation: To establish a mechanism-based rescue of impaired pathological fracture healing by targeting specific pro-inflammatory kinases in vivo. We expect to establish a novel therapeutic paradigm for devastating patients suffering from cancer and pathological fractures.

NIH Research Projects · FY 2026 · 2024-02

The use of complex, multi-component interventions (CMCIs) is an increasingly important aspect of cardiovascular disease prevention, screening, and treatment. For example, this application’s co-Investigator Longenecker is the principal investigator of a CMCI trial nearing completion aimed at improving blood pressure control, EXTRA-CVD (XCVD), consisting of 4 components: 1. Nurse-led care coordination, 2. Nurse-managed medication protocols and adherence counseling 3. Home blood pressure (BP) monitoring, and 4. Electronic health record (EHR) support tools, to be compared with generic prevention education. Each component has a “dose”, e.g. number of days/week home BP should be recorded and reported, and number and duration of adherence counseling sessions. Implementation scientists, such as those conducting this trial, discuss the tension between fidelity to the original intervention protocol, and the need for tailoring, tweaking and adaptation, perhaps contextually driven, i.e. varying by facility size, composition of the provider workforce, or health status of the patient population served. Under our innovative Learn-As-you-GO (LAGO) design, as the trial evolves, the doses of these intervention components are adapted at pre-specified stages to maximize cost-effectiveness and reduce the ultimate risk of trial failure by achieving pre-specified statistical power, while at the same time preserving the nominal size of the hypothesis test for the overall intervention package effect and attaining a pre-planned study outcome goal, such as attainment of 80% of patients under blood pressure control. Because each intervention component is associated with different costs and effectiveness, it is difficult to specify the optimal intervention package, that is, the optimal intervention component “doses” along with the components themselves, before launching a trial, as standard methods require. LAGO designs for continuous outcomes, such as changes in blood pressure (mmHg) or cholesterol levels (mg/dL) as in XCVD; for repeated binary outcomes, such as per visit hypertension control; and which account for clustering of outcome rates within centers are not available. In this project, following on our 2021 Annals of Statistics publication establishing fundaments for logistic regression analysis of a single binary outcome in non-clustered data, we will derive the mathematical theory for LAGO designs for clustered binary and continuous repeated measures data, and compare the LAGO design to its standard non-adaptive factorial and two-armed alternatives where the intervention is fixed before the study commences. Once a LAGO study concludes, LAGO provides a means to design interventions for new centers, scaling up and out, subject to the same class of goals, perhaps contextually dependent. Methods will be applied to XCVD and PULESA-Uganda, another active CMCI trial led by co-Investigator Longenecker for which contact PI Spiegelman serves as study statistician, allowing for this trial to serve as a living laboratory for LAGO methods development. User-friendly, publicly available software will be produced and disseminated, to facilitate use of these designs by practitioners.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Mammals have evolved several antibody isotypes capable of engaging distinct response pathways upon subsequent recognition of antigen. Immunoglobulin E (IgE) is an antibody isotype highly specialized to rapidly and potently activate Type II effector cells. The deleterious role of IgE in allergic disease has been well- described in the literature extending back to the early 20th century. However, the roles of IgE in homeostasis and in host protection from disease remain poorly understood. One approach to understanding the functional role(s) of antibody isotypes is through identifying targeted antigens. Reactivity profiling has been performed for all antibody isotypes except for IgE and has been informative in establishing the tissue sites, effector pathways, and functional consequences of antigen recognition by specific antibody isotypes. The antigenic targets of IgE have not been broadly characterized to date largely due to the difficulty in obtaining sufficient antibody quantities for testing. We created a genetic tool to both extend the half-life of IgE and to facilitate IgE purification, enabling high-throughput reactivity screening. Preliminary data for this proposal showed that during homeostasis and after infectious challenge, IgE and IgG recognized distinct sets of antigens. Some reactivities unique to the IgE antibody pool were conserved under all tested conditions, suggesting that these antigen:antibody interactions are an important component of fundamental IgE biology. Additional data demonstrated that homeostatic IgE had a profound influence on the host response to acute inflammatory challenge, and that this influence was antigen- dependent. We propose to fully resolve IgE reactivities in homeostasis (Aim 1), in relevant genetic and environmental contexts (Aim 2), and ultimately to contextualize IgE effector function(s) within the host response to acute inflammatory perturbation (Aim 3).

NIH Research Projects · FY 2026 · 2024-02

Summary/Abstract Chorioamnionitis - inflammation of the fetal membranes (FM) - is characterized by neutrophil infiltration and is a major risk factor for preterm birth. Even in the absence of prematurity, chorioamnionitis can be detrimental to the fetus. Despite a strong association between bacterial infection, chorioamnionitis, and preterm birth, the mechanisms involved are not fully understood. Through expression of the innate immune pattern recognition receptors (PRR), Toll-like receptors (TLRs) and Nod-like receptors (NLRs), FMs have strategies to evade and protect against infection. However, depending upon the nature of signaling and regulation, these protective immune mechanisms may create an inflammatory milieu that can contribute to pathology. In particular, IL-8 is a major neutrophil chemoattractant and inflammasome-mediated IL-1b is a major inducer of tissue injury and mediator of preterm birth. We have found that the chorionic compartment is the primary site of FM IL-8 and IL- 1b production in response to bacterial lipopolysaccharide (LPS), peptidoglycan (PDG), and muramyl dipeptide (MDP) which activate TLR4, TLR2, and Nod2, respectively. While TLRs and NLRs can directly activate signaling pathways leading to inflammatory cytokine/chemokine production, there is the potential for far more complex modulation, regulation, and fine-tuning of these processes and the type of responses generated, particularly for IL-1b. This grant focusses on the requirement of two or more PRRs to be activated sequentially in FMs exposed to bacterial triggers via novel intermediates. A non-classical family of microRNAs (miRs) activate the ssRNA sensors, TLR7 and TLR8, to elicit an inflammatory response. These miRs can also be carried in exosomes and delivered to TLR7 or TLR8 in target cells. Our preliminary data supports the concept that TLR8-activating miR-146a-3p may acts as a novel intermediate signal that drives FM chemotactic and inflammatory responses to bacterial TLR and NLR agonists. We also have preliminary data demonstrating that FM-derived exosomes containing TLR8-activating miRs trigger neutrophil activation and release of neutrophil extracellular traps. Finally, we found that FM tissue and circulating exosomal TLR8-activating miR-146a-3p is elevated in women with preterm birth. Based on this, our central hypothesis is that TLR8-activating miRs mediate FM chemotactic IL-8 and inflammasome-mediated inflammatory IL-1b in response to bacterial triggers, and through their release and delivery via exosomes active maternal neutrophils. This leads to inflammation at the maternal-fetal interface, increasing the risk for chorioamnionitis. To test this, our specific aims are to determine if: Aim 1. TLR8-activating miRs mediate a FM chemotactic IL-8 response after exposure to bacterial triggers. Aim 2. TLR8-activating miRs contribute to FM inflammasome activation and inflammatory IL-1b production. Aim 3. FM exosomes containing TLR8-activating miRs induce neutrophil activation.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Olfaction plays a critical role in animal survival, enabling the detection of food sources, dangers, and potential mates. The remarkable plasticity of the olfactory system allows for the modification of responses to cues based on various internal states. However, the precise mechanisms underlying olfactory plasticity, particularly at the level of peripheral sensory neuronal activity, remain poorly understood. In this research proposal, we aim to investigate the contribution of long non-coding RNAs (lncRNAs) and lncRNA-encoded micropeptides in modulating olfactory receptor neurons (ORNs) and other olfactory functions. LncRNAs are transcripts longer than 200 nucleotides that lack an open reading frame longer than 100 codons. Various lncRNAs have been implicated in neural development, function, and diseases. While lncRNAs are traditionally considered non-coding, certain lncRNAs encode micropeptides, which contribute to diverse biological processes. Despite their abundance and potential importance, the functions of lncRNAs and their encoded micropeptides in the nervous system, particularly in olfaction, remain largely unexplored. To address this knowledge gap, we will capitalize on the well-characterized olfactory system of the fruit fly. This system offers several advantages, including numerical simplicity, well-defined neurons that drive complex behaviors, and the availability of powerful genetic tools. We recently generated a comprehensive survey of lncRNAs in the main fly olfactory organ that demonstrated the diversity and expression patterns of lncRNAs in the olfactory system and set the stage for investigating their functional roles and their impact on sensory behaviors. Through a multidisciplinary approach that includes genetic, electrophysiological, behavioral, and molecular assays, we will test the hypothesis that lncRNAs and their micropeptides contribute to olfactory modulation and function. Our initial focus will be on the lncRNA ANRUS (Antennal RNA Upregulated by Starvation) and its encoded micropeptide. We will functionally characterize ANRUS and test whether it contributes to olfactory modulation. During the R00 phase, we will investigate the unexpected regulation of ANRUS levels by a food odor, ethyl acetate. Finally, we propose to expand our investigation by characterizing the micropeptidome of the antenna, a first step toward exploring the regulatory roles of these micropeptides in olfaction and gaining a deeper understanding of the mechanisms governing olfactory plasticity and function. Dr. Talross will benefit from outstanding support from her mentoring team at Yale University, which will facilitate her journey towards independence. The proposed objectives in this proposal are carefully designed to equip Dr. Talross with the necessary skills and experience to secure an Assistant Professor position and pursue R01 funding as an independent investigator.

NIH Research Projects · FY 2025 · 2024-02

Summary Chimeric Antigen Receptor (CAR)-T cell therapies have achieved unprecedented success in treating blood cancers that are resistant to traditional radiation or chemotherapies. However, the efficacy of CAR-T therapy to solid tumors remains limited. Major challenges include poor infiltration of T cells into tumors, exhaustion and low persistence of T cells under a hostile tumor environment. To tackle these problems, we aim to seek alternative cell carriers for CAR. Mast cells present several advantages in targeting solid tumors: they reside in tumor tissues and can live up to years. They can repetitively kill target cells without exhaustion. They also release cytokines that can recruit other effector cells to kill cancer. In this project, we will design CARs that specifically activate mast cells to kill solid tumors, using melanoma and colon cancer as models. Our efforts are expected to lay the scientific foundation for a brand-new cell therapy, and provide rationales and generate reagents for moving towards further pre-clinical and clinical studies.

NIH Research Projects · FY 2026 · 2024-02

Preeclampsia (PE) is a syndrome of new hypertension (HTN) with organ damage that occurs in 3-8% of pregnancies and is a leading cause of maternal mortality. Women who survive PE have a substantially increased risk of future HTN, heart attack and stroke by unknown mechanisms. These women have enhanced blood pressure and vasoconstriction responses to HTN stress that persists months to years after PE. In male mice, T cells are necessary for hypertension and effector memory T cells contribute to exacerbated responses to repetitive hypertensive stresses. To explore mechanisms driving post-PE HTN, we modified two models of PE; one is induced by overexpression of the anti-angiogenic soluble VEGF receptor 1 (sFlt1) during pregnancy and the other is induced by hypoxia during pregnancy. I confirmed that both models cause increased sFlt1 and other features of PE seen in humans. Preliminary data in the sFlt1 model reveals that despite post-partum sFlt1 levels and blood pressure normalizing: (1) post-partum microvascular structure/function abnormalities persist; (2) post-partum HTN stimuli results in an exacerbated blood pressure response, microvascular vasoconstriction and microvascular expression of the T- cell chemokine, CCL5; and (3) kidney effector memory T cells are significantly increased after HTN stimuli. Thus, I propose to test the hypothesis that experimental PE causes long-term T cell- mediated changes in the microvasculature and kidney that increase sensitivity to post-partum HTN stimuli. Aim 1 will examine if T cells are necessary for persistent vascular remodeling and dysfunction after PE. T cell populations, migration and cytokine expression will be measured during and after PE and in response to hypertensive stimuli. T cells will then be depleted and blood pressure and vascular structure/function analyzed. Aim 2 will determine if adoptive transfer of T cells exposed to PE is sufficient to induce the vascular and kidney changes associated with post-PE HTN. Aim 3 will test the specific role of memory T cells in exacerbating the response to hypertensive stimuli after PE. Completion of the aims will provide new insight into the mechanism driving the substantial increase in HTN risk after PE, thereby supporting the NIH mission to improve maternal health. The proposal will also allow me to gain new expertise in HTN diseases of pregnancy and foundational immunology techniques. The mentoring team assembled on this application, with expertise in cardiovascular immunology, nephrology, pregnancy and vascular biology, the environment at Tufts Medical Center and Tufts University and the training plan proposed will further strengthen my ability to become an independent investigator studying mechanisms driving heart diseases in women.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY The high rate of unplanned pregnancies suggests that currently available contraceptive methods are not effectively meeting the needs of women. In addition, contraceptive options for men are limited to vasectomy and condoms, leaving a significant unmet need for contraception. Our long-term goal is to develop a non-steroidal, effective contraceptive that provides a more comprehensive approach to birth control. We propose that the sperm-specific CatSper calcium (Ca2+) channel is an ideal target for the development of such a new class of contraceptives that has no negative side effects in either men or women, as the CatSper channel is a validated target required for sperm capacitation and male fertility in both mice and humans. Drug inhibition of CatSper at the post-testicular and pre-fertilization stages would work without off-target side effects due to its post-meiotic expression in male germ cells and functional divergence from other calcium channels. The recently solved struc- tures of CatSper highlight its high accessibility in the cell membrane, allowing the study of the mechanism of action and reversible contraceptives. However, the inability to reconstitute the channel in vitro has been a bot- tleneck in the development of drugs that directly target the CatSper channel. We have recently overcome this hurdle by creating chimeric CatSper channels that heterologously express functional channels. Using this new tool, the overall goal here is to ultimately develop CatSper modulators that inhibit human sperm function. To this end, in R61 phase, we will perform in-depth biophysical and functional characterization of these novel chimeric channels and do molecular dynamics studies to gain new insights (Aim 1) and develop the necessary assays for primary and secondary screening that measure CatSper activity in high-throughput modes (Aim 2). In the R33 phase, we will perform high-throughput screening for CatSper inhib- itors as well as virtual screening (Aim 3), profile the identified hits, establish preliminary structure-activity rela- tionships and perform the secondary screening (Aim 4), and test the selected compounds on human sperm function (Aim 5). In the immediate term, successful completion of these aims will provide small molecule hits for human CatSper that can be used for iterative lead generation. In the long term, the leads and knowledge gener- ated will ultimately lead to the development of an innovative class of contraceptives targeting CatSper and sperm capacitation with a mechanisms of action foundation.