Tufts University Medford

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract: In the human endometrium, the acute patterns of immune infiltration and expression of inflammatory signals are found across the menstrual and are needed for maintaining reproductive function including implantation, menstruation and regeneration. Regulation of these timely events is primarily driven by fluctuations in estrogen and progesterone signaling, however, the regulatory mechanisms that govern resolution of these inflammatory signals remain unknown. The resolution of inflammation is a coordinated and bioactive process aimed at restoring tissue integrity and function after acute inflammatory insults. Poor resolution of inflammation is a mechanism in the pathogenesis of many chronic inflammatory diseases, like endometriosis. A central question in endometrial biology that remains unanswered is how the resolution of inflammatory processes is regulated across menstrual health. We will investigate the role of specialized pro-resolving mediators (SPMs), a class of lipid-derived molecules that act as potent endogenous immunoresolvents that orchestrate the resolution of inflammation and immune function, in menstrual health. We hypothesize that fluctuations in estrogen and progesterone regulate the inflammatory responses of the endometrium via SPM biosynthesis and dysregulation of SPM signaling drives the pathogenesis of endometriosis. One significant barrier to progress in this area is the lack of humanized models that can temporally and mechanistically parse the immune- endocrine interactions in human endometrial health. We deploy innovative multi-cellular organoid in vitro models and computational analysis of the human endometrium datasets to address three key knowledge gaps: 1) is the resolution of inflammation is regulated by sex hormones, 2) how do changes in inflammatory networks positive and negatively impact reproductive processes and 3) can these pathways be targeted as a non-hormonal treatment for endometriosis. In this line, we will use this framework to evaluate how extrinsic factors, like dietary- derived fatty acids impact subsequent inflammatory responses. A deeper understanding of the lipidome and the mediators that regulate the resolution of inflammation is necessary to advance our understanding of fundamental reproductive and inflammatory events. A primary goal for this proposal period is to deliver a comprehensive and mechanistic atlas of SPM signaling pathways in endometrial health to interrogate how inflammatory signals are impact reproductive function. In the end, we will gain fundamental insights into the sex hormone and immunological origins of reproductive function, setting the stage for understanding fundamental biological and inflammatory processes including menstrual bleeding, regeneration and nociceptive pain signaling. Ultimately, findings from this research program will open new avenues for using SPM as novel therapeutic target.

NIH Research Projects · FY 2026 · 2026-05

Project Summary The focus of my lab is to understand the mechanisms of genome instability caused by error-prone DNA repair and damage tolerance mechanisms. Specifically, we are interested in the roles of translesion DNA polymerases in double-strand break repair and lesion bypass. Recent studies have shown that cancer cells upregulate the expression of translesion polymerases and that they can become addicted to them, providing potential therapeutic targets. However, because most of these studies utilize cells grown in culture, tissue- and development-specific context that may be important for understanding treatment efficacy is often lost. To address this, we have pioneered the use of a Drosophila model to study DNA damage tolerance and error- prone double-strand break repair pathways. We made the important discovery that DNA polymerase theta is a key protein involved in alternative end-joining repair of double-strand breaks, which prevents the creation of large deletions when homologous recombination repair is impaired. We recently demonstrated that highly proliferative tissues in Drosophila rely heavily on translesion synthesis (TLS) bypass to complete replication and prevent genomic catastrophe following alkylation damage. We identified at least two ways that the REV1 translesion polymerase scaffold protein promotes bypass of alkylation damage, and we have shown that the SLX1/4 structure-specific nuclease becomes important for tolerance in a TLS-deficient background. Using a forward genetic screen for mutations that impair survival specifically when TLS is compromised, we have identified several novel genes that promote tolerance of both exogenous and endogenous DNA damage. Building on these studies, we plan to advance our research program in several directions. First, we will further determine the contribution of template switching and fork reversal mechanisms to damage tolerance in Drosophila, as these represent critical backup pathways that could contribute to therapeutic resistance when TLS is impaired. Second, we will use genetic, molecular, and biochemical assays to precisely define how the genes identified from our screen act to promote replication past DNA lesions, prioritizing those that have not previously been associated with damage tolerance. Third, we will investigate the mechanistic basis behind our observation that haploinsufficiency of the BRCA2 gene causes synthetic lethality when key replication and repair proteins are mutated, focusing on bypass of endogenous DNA damage. We will utilize chromosome spreads, damage visualization techniques, and whole-genome sequencing to determine the consequences of loss of key tolerance mechanisms on genome integrity. Together, these studies will provide important insight into the ways that cells prioritize various damage tolerance and repair strategies in the context of developing tissues, and the consequences that these strategies have on mutagenesis and chromosome instability.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY While wound healing processes are vital for successful organismal tissue repair, failure to turn off these mechanisms lead to the excessive accumulation of the extracellular matrix (ECM) and the development of fibrosis. Although recent research is beginning to illuminate the circumstances that allow for fibrosis resolution, most fibrotic conditions remain unresolved; resulting in organ failure or a predisposition to cancer. While the triggers for fibrotic diseases fall within a handful of categories, including viral/bacterial infection, tissue damage, and chemical insults, all induce the sustained activation of mesenchymal cells into myofibroblasts. Besides transforming growth factor β (TGF-β), which is a major activator of fibroblasts, the ECM also has the ability to alter a fibroblast’s activation state. We and others have demonstrated that this activated phenotype can persist despite the cell’s removal from fibrotic tissues, suggesting that fibroblasts have an “epigenetic memory” acquired during activation and retained thereafter. However, the molecular details underlying epigenetic regulation during myofibroblastic activation and whether these details are universal despite the activation trigger is unknown. One potential mechanism of myofibroblast regulation is through the ECM-dependent expression of pro- fibrotic genes. Gene expression is a tightly regulated process that requires chromatin remodeling, binding of transcriptional activators, and recruitment of RNA polymerase to initiate transcription. As such, the structural reorganization of the chromatin plays a large role in the temporal regulation and tissue specificity of gene expression. Brahma-related gene 1 (BRG1) is a central catalytic ATP-subunit of the BAF (BRG1/BRM1- associated factor) complex which works to drive chromatin accessibility via nucleosome eviction. BRG1 has also been shown to regulate ECM gene expression in both healthy and virally-induced fibrotic contexts. Moreover, preliminary data found that BRG1-deficient pancreatic fibroblasts lost the expression of a key functional regulator, Netrin G1. Taken together, this suggests a role of BRG1 in regulating myofibroblast pro-fibrotic genes. The overarching goal of this proposal is to test the hypothesis that the ECM and fibrosis-inducing viruses alter fibroblasts’ chromatin landscape in a BRG1-dependent manner and contribute to the epigenetic memory that underlies myofibroblastic function. To test this hypothesis, Aim 1 will first investigate BRG1’s involvement in pancreatic fibroblast activation in vitro and whether this is regulated by ECM-mediated signaling. In Aim 2, experiments will focus on the role of BRG1 in regulating disease formation by using an in vivo pancreatitis mouse model. Finally, Aim 3 will build on these lessons and investigate the mechanisms by which fibrosis-inducing viruses cause myofibroblast activation, beginning with the frequent human pathogen, Influenza A virus. By using a novel perspective to understanding fibrosis, this research provides insights that will advance cell biology, epigenetics, and virology, as well as reveal how the ECM/viruses create a pro-fibrotic state.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY / ABSTRACT Optical finger pulse-oximetry does not fully account for individual-specific anatomy or hemodynamics, leading to possible inequalities in care due to skin-tone biases. Specifically, there is growing evidence that conventional pulse-oximetry methods have a positive bias for dark versus lighter-skinned patients, mean- ing an incorrectly high oxygen saturation will be recorded for dark-skinned patients. This type of bias is dangerous since it may lead to occult hypoxemia, where low blood oxygen levels are missed, and the proper treatment isn’t prescribed. This research project aims to pioneer a noninvasive and auto-calibrated pulse-oximetry method that more accurately and unbiasedly determines a person’s arterial oxygen sat- uration than conventional pulse-oximetry. The project will be achieved by completing the project aims of theoretical development of the proposed person-specific pulse-oximetry method, device prototyping and fabrication, and finally, device testing in a clinical environment. Theoretical development will entail two activities: developing an inverse model to recover person-specific finger optical properties from an auto-calibrated dual-ratio measurement and spatial mapping of pulsatile hemodynamics in the finger to determine which tissue should be targeted for the proposed pulse-oximetry technique. Then, a custom wearable device will be designed and fabricated to implement this person-specific pulse-oximetry method. Finally, the device and method will be tested in the clinic, where gold-standard measurements of arterial saturation are routinely made so that a gold-standard comparison can be made with the proposed pulse- oximetry method. The positive impact of this technique will be an equitable and non-biased noninvasive assessment of one’s arterial oxygen saturation, a ubiquitous and clinically relevant measure of oxygen perfusion. Throughout the work on this project, the principal investigator, Giles Blaney, PhD, will complete career development activities to enable him to be a successful faculty candidate. The potential for career development in this project is significant. By undertaking device prototyping and fabrication, Dr. Blaney will gain valuable electronic device design and fabrication skills, while during the theoretical development stage, Dr. Blaney will gain experience in mathematical inverse modeling. Additionally, this project will also serve to develop Dr. Blaney’s writing and mentoring skills. Overall, this project aims to address a bias in current pulse-oximetry methods and foster the career development of the principal investigator, Dr. Blaney.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The Ding laboratory focuses on advancing drug design by developing computational methods that integrate molecular simulations with machine learning. We implement our innovative methods into open-source software, accompanied by comprehensive documentation and tutorials, making them accessible for both practical drug discovery programs and further methodological research. We believe that the full potential of computational techniques in drug design remains largely untapped, especially considering the rapid advancements in computing power and breakthroughs in machine learning and modeling technologies. Our long-term goal is to harness these advances to create transformative computational tools to accelerate drug discovery. In the next five years, we will focus on advancing methods for computing protein-ligand binding free energies, a key metric for drug design. Current techniques, such as relative binding free energy (RBFE) methods, have proven valuable in optimizing lead compounds. However, they are limited in scope, as they can only compute changes in binding free energy caused by small modifications on a reference compound. This restricts their use in exploring more chemically diverse compounds, which are often critical for early stages of drug discoveries. To address these limitations, we aim to advance methods for computing absolute binding free energies (ABFE), which allow for accurate binding affinity calculations without requiring a reference compound. While ABFE methods hold great promise, current approaches are either computationally expensive or inaccurate for wide practical use. Our goal is to create ABFE methods that overcome these challenges, making them both accurate and efficient for broad application in drug design. In parallel, we recognize that drug discovery is an iterative process in which computational predictions and experimental data accumulate over time, offering valuable insights into target protein and ligand interactions. However, current methods fail to fully leverage this expanding body of knowledge in a drug design program. To overcome this limitation, we will develop a Bayesian framework that incorporates prior information from both simulations and experiments into free energy calculations. This approach will enhance the precision, accuracy, and efficiency of these calculations, improving both relative and absolute free energy methods. During the development of these algorithms, we will rigorously evaluate their performance through benchmarking against existing datasets and collaborating with experimental groups in drug design projects. Additionally, we will test our method’s performance prospectively by participating in blind challenges. All new developments will be made available through open-source software, maximizing their impact on the broader drug discovery communities.

NIH Research Projects · FY 2025 · 2025-09

Objectives: Our study will characterize the immediate and cumulative health impacts associated with extreme weather events and will identify the key determinants that make communities vulnerable or resilient to these impacts. It will do so by focusing on communities within the Gulf and southeastern Atlantic United States (US) coastal and adjacent regions, where the adverse consequences of such events are particularly severe. Our study has three primary aims: to (1) perform a comprehensive vulnerability assessment that describes the hazards, as the time, location, magnitude and severity of each extreme weather event, and community vulnerability, based on the infrastructural, environmental, and socio-economic characteristics of each ZIP code within our study region, (2) assess the health impacts from extreme weather events and to identify community factors that modify these impacts, and (3) characterize the speed and degree to which the health of communities recover post-extreme storm events and examine whether overall and component-specific vulnerability indicators predict health recovery class membership. Approach: Our approach is inter-disciplinary, leveraging expertise from civil and environmental engineering, epidemiology, and biostatistics. Using data from 1990 to 2021, we will develop a multi-dimensional vulnerability framework to (1) assess the hazards posed by extreme weather events using data on biophysical factors and probabilistic modeling, (2) characterize the vulnerability of communities to these events using machine learning techniques that incorporate information on the infrastructural, socio-economic, and environmental characteristics of these communities, and (3) identify clusters of communities that share similar vulnerability profiles using k-mean and hierarchical clustering techniques. We will examine health risks and characterize community recovery profiles post-extreme weather events and their modification by event type and vulnerability indicators. Importantly, our examination will focus on all Medicare and Medicaid beneficiaries living in the southeastern US. We will do so using individual-specific, all-cause mortality and cause-specific hospital admissions data for these beneficiaries and using epidemiologic models, such as mixed effect logistic regression models with distributed lag effects and linear segmented regression models. Expected Results: Our study is expected to yield new evidence and critical insights into the complex interplay of factors that influence the vulnerability and health of coastal communities facing extreme weather events. This knowledge will inform strategies related to emergency planning, healthy recovery efforts, and the enhancement of resilience among these communities.

NIH Research Projects · FY 2025 · 2025-09

Biological networks are powerful resources for the discovery of genes and genetic modules. For the classical physical protein-protein interaction network, as well as for other types of pairwise association data between genes or proteins that is organized according to homophilic principles, diffusion-based low dimensional network embedding methods have proved quite powerful. While at a coarse scale, genetic interaction networks, built from high-throughput epistasis experiments, also display some homophily in their organization, at a fine scale, they display very different graph-theoretic structure that can be leveraged to find mechanisms of redundancy and fault tolerance among biological pathways active in the cell. However, the graph theoretic toolbox to analyze this fine-grained structure in genetic interaction networks is still underdeveloped, compared with algorithms developed for analysis of the purely homophilic biological networks. Inspired by some of the tools and techniques used in graph theory to study vertex cuts in networks, we propose to develop a more formal framework for categorizing the patterns of resilience and redundant pathway mechanisms in genetic interaction networks, and new algorithms for discovering genes involved in compensatory pathways. We will use our mathematical framework to computationally make phenotype predictions for the results of gene knockout experiments with either unseen gene combinations or in unseen environmental conditions. We will validate some of our predictions focusing on alternative pathways for DNA replication and repair in Saccharomyces cerevisiae (baker's yeast). Since DNA replication and repair are crucial pathways for cancer cell survival, identification of genes in those subnetworks could reveal new cancer therapy targets. Discovery of new genes is important for a basic understanding of cell functioning and to elucidate the cause of inherited genetic disease. RELEVANCE (See instructions): Saccharomyces cerevisiae is a foundational model system for studying mechanisms of resilience and fault tolerance in the Eukaryotic cell, and the pathways for DNA replication and repair are sufficiently evolutionarily conserved to make insights in these model systems relevant for understanding human diseases that exhibit genome instability. Methods to target compensatory pathways are also an active area of drug design, including in the search for personalized therapies customized by tumor in cancer.

NIH Research Projects · FY 2025 · 2025-09

Abstract Studying a cell’s ability to sense and respond to mechanical cues has emerged as a field unto itself over the last several decades and is now appreciated by engineers and biologists alike. For most cell types investigated thus far, when cultured on soft substrates they have slower growth rates, smaller spread areas, lower traction, and lower migration rates than when cultured on stiff substrates. However, there is a critical gap in the literature. These prior studies, with two notable exceptions, have all taken cell lines previously propagated on plastic surfaces and assayed cell response to substrate stiffness over the course of a few days to a week. These studies, which have been seminal and established the field of cell mechanosensing, are limited to short term phenotypic responses. Taking an intellectual leap, this proposal will test a novel concept of cellular evolution driven by substrate stiffness. We hypothesize that the mechanical properties of the substrate will select for variants fit for growth on soft or stiff substrates out of a genetically heterogeneous population of cells, just as drug treatments select for genetically resistant cells. We will test this hypothesis by performing sustained culture of cells on soft and hard hydrogels. Across two aims we will quantify how cell phenotypes (growth, motility, metastasis) evolve as cells are cultured for increasing lengths of time on soft and stiff hydrogels, sequence genomic changes during this mechano-evolution, and alter the link between mechano-evolution and phenotypic consequences through targeting mechanosensors.

NIH Research Projects · FY 2026 · 2025-06

Project Summary/Abstract The escalating presence of microplastics (MPs) and nanoplastics (NPs) in the environment raises alarms about their potential impact on both ecological systems and human health. Notably, these particles are often found in the human gastrointestinal tract, yet a comprehensive understanding of their health effects remains elusive. In our lab, we excel in establishing three-dimensional (3D) mini-intestine models sourced from human intestinal organoids, allowing us to simulate both healthy and diseased intestines. Leveraging these capabilities, recent studies in our lab using organoid-derived monolayers—both with and without M cells—have identified clear interactions between human intestinal organoids and polystyrene MPs/NPs. Crucially, we observed that the dose, size of the particles, and the presence of M cells significantly modulate immune responses. Our ongoing research endeavors aim to expand on these findings by synergizing our specialized in vitro 3D human intestinal models with comprehensive in vivo mouse studies. By examining the implications of exposure to various environmentally relevant MPs and NPs across different biological hierarchies—from molecular to cellular to individual levels—we aim to provide a comprehensive understanding of the potential health consequences. We hypothesize that environmentally relevant MPs and NPs disrupt gut homeostasis, exacerbate inflammation in intestines already compromised, with the intensity governed by their attributes and exposure duration, and that M cells play a crucial role in mediating these immune responses. To test the hypothesis, we proposed the following aims. Aim 1 focuses on assessing the acute and chronic effects of MPs and NPs on intestinal health. Using patient-derived, 3D bioengineered human and mouse intestinal models, we will explore how different particle characteristics and exposure durations influence gut health. This will involve comparing the effects of different plastic particles on in vitro 3D human and mouse models, helping us differentiate species-specific responses. Aim 2 investigates the influence of MPs and NPs on inflamed intestines. Our hypothesis posits that these particles exacerbate inflammation in already inflamed gut environments. By using an inflamed variant of our 3D human intestinal model, we will track inflammation markers and examine how plastic particles affect tissue integrity and microbial balance. Findings from the human models will be corroborated through whole- organ examinations in inflamed mouse models, ensuring a holistic understanding of systemic reactions. Aim 3 seeks to unravel the specific role that Microfold (M) cells play when interacting with MPs and NPs. We postulate that these cells can amplify the gut's response to plastic particles, potentially driving inflammation due to their unique properties. This aim will involve creating multiple model variations to explore the apoptotic effects of particles on M cells and their subsequent impact on inflammation. Our project deciphers the multi- dimensional impacts and underlying mechanisms of MPs and NPs on intestinal health, thereby informing future clinical and environmental strategies.

NIH Research Projects · FY 2026 · 2025-06

Protein post-translational modification (PTM) networks are tremendously promising, but vast, frontiers for drug discovery. It is therefore essential to learn how specific PTMs are integrated into signaling networks in living cells. To address this need, the Scheck lab pioneers new chemical biology tools that will provide critical insight about PTM networks in living cells. Our focus is on PTMs like glycation and ubiquitination, which have been particularly difficult to study using traditional tools that inhibit or profile specific enzyme activities through genetic knockout or pharmacologic inhibition. Our novel methods will unlock previously unattainable information that will be used to address longstanding questions about the role of glycation, ubiquitination—and their interplay—in diabetes, cancer, inflammation, neurodegenerative disease, and other age-related disorders. Learning how these signals are integrated into cellular signaling processes will provide access to new targets for preventing or treating numerous diseases. This MIRA project describes chemical strategies that rely on our knowledge of PTM chemistry and mechanism, making us uniquely suited to develop needed methods to track specific glycation or ubiquitination events in living cells. One series of projects builds on our significant published and unpublished work that has uncovered the chemical and molecular features that underpin selective glycation, enabling us to create a novel tool (called dialAGE) that predictably modulates site-specific glycation events in living cells. The proposed studies include the use of deuterated methylglyoxal probes to differentiate previously indistinguishable AGE isomers using unbiased quantitative proteomics. We will also evaluate how differential ubiquitin glycation influences the global ubiquitinome and learn how it influences protein turnover through proteasomal or autophagic pathways. We will also create a new set of dialAGE tools to manipulate histone glycation, enabling studies that will reveal how glycation influences chromatin compaction, PTM crosstalk, and in vitro transcription. Another series of projects builds on a new tool we recently reported, called targeted Charging of Ubiquitin to E2 (tCUbE), that tracks the fate of ubiquitin through its sequential E1-E2-E3 cascade all the way to its ultimate target. The proposed studies will optimize tCUbE as an enabling technology that can be broadly used to profile each of the 38 known human E2s. We will also use tCUbE to interrogate specific E2s, such as UBE2L3, which we hypothesize to exhibit multiple activities that differentially engage disease-relevant signaling pathways. Uncovering new E2-substrate interactions or cascades will enable discovery of targeted degradation or stabilization therapies to disrupt or rewire specific steps within the UPS. Together, this work will uncover direct links between glycation, ubiquitination, and critical intracellular processes, including proteasomal degradation, mitophagy, and chromatin remodeling. These studies will dramatically improve our understanding of glycation and ubiquitination, and will have an immediate impact on our appreciation for how they influence human health, aging, and disease.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY/ABSTRACT During development, coordinated formation of key structures in the nervous and musculoskeletal systems are mediated through a specific "HOX code" that confers positional identity and contributes to cell subtype diversification and connectivity. To understand human nervous system diversity and subtype-specific neurodegenerative pathologies, it's essential to efficiently derive these region-specific cells from human pluripotent stem cells (hPSCs) in vitro. Yet, current small molecule-based approaches fall short of achieving desired efficiency while transcription factor (TF) overexpression methods fail to capture regional identity. Therefore, our goal is to develop a hybrid strategy to produce high-purity, region-specific cell populations. This project builds upon a recent innovation in our lab: a modular directed differentiation platform for generating spinal neurons from anywhere along the rostrocaudal or dorsoventral axes. To do this we go through intermediate neuromesodermal progenitors (NMPs), bipotent axial stem cells that are the first to acquire a distinct HOX code before diverging into mesodermal, neural tube, and neural crest progeny. Here we focus on expanding this platform to oligodendrocytes (OLs) and neural crest progenitors (NCPs), which share a common TF regulator, SOX10. We hypothesize that inducing SOX10 expression at key stages of differentiation will enable rapid generation of both lineages from the same region-specific NMP protocol. In Aim 1 we employ a TET-inducible SOX10 strategy to rapidly differentiate region-specific NCPs. In Aim 2, we seek to optimize the directed differentiation of region-specific OLs and determine whether the TET-inducible SOX10 strategy speeds differentiation and maturation to a myelinating phenotype. We will also determine whether the hybrid method enables generation of novel dorsal vs. ventral OL subtypes, which have not been generated from hPSCs previously. Thus, our project aims to define a next-generation system for producing region-specific cells that can be implemented using other TFs to create a translational region-specific toolbox. Future investigations may leverage this toolbox to advance our understanding of cell fate decision making, develop personalized cell therapies, or integrate into more accurate in vitro models of neurodegeneration, demyelination, and chronic pain. Ultimately we anticipate that this broad applicability will lay the groundwork for discovering novel therapeutic strategies for regenerative medicine.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY/ABSTRACT Liquid venous blood is the gold standard for the majority of clinical assays, but it is unrealistic to expect it can be collected at home or in remote locations due to the need for trained phlebotomists and the costs and logistics related to cold chain transport. Approaches that enable patient-centric microsampling—where blood that is self- collected by fingerstick can be sent ambiently through the mail—have the potential to address challenges with specimen transport. However, the development of these technologies, typified by the century-old dried blood spot (DBS) card, has not kept pace with the clinical need or emerging capabilities of telemedicine. The ability to maximize the diagnostic test menu available to patients fundamentally relies on the successful transport of dried blood or plasma to clinical laboratories. To make reliable clinical decisions, enable diagnoses, and inform treatment decisions, there is an outstanding need for functional patient-centric devices that stabilize specimens and enable measurements that are consistent with venous blood. Any solution must support two unique sets of users: (i) Patients who desire access to routine tests for chronic conditions that would otherwise require them to travel to receive care, and tests that provide personalized insight into their health status. (ii) Laboratories that require high quality samples that do not introduce unpredictable sources of error into the quantitative measurements that will be used to inform healthcare decisions, and processes that do not disrupt their current analytical workflows. Previously, we demonstrated that patterning DBS cardstocks can create metered collection zones for microsampled dried blood and plasma that support measurements of a wide range of analytes that are equivalent to those made using liquid blood. Using our expertise in paper microfluidic devices, we propose the development of paper-based microsampling devices that are designed to further close the gap between current laboratory testing capabilities and ongoing patient needs by providing stable and metered samples of dried blood and plasma to existing clinical workflows. Innovations in patient-centric, microsampling technologies will reduce global disparities to access in healthcare, provide increased agency to patients desiring more information about their health status, and support ongoing advances in telemedicine. We anticipate that the capabilities demonstrated by our patterned dried blood and dried plasma spot cards will serve these needs and facilitate improvements in patient care.

NIH Research Projects · FY 2026 · 2025-02

Summary: Heart disease is the leading cause of death in the United States and there are currently over 5 million people in the U.S. affected by heart failure (HF). One potential therapeutic that is currently undergoing Phase I clinical trials is the injection of solubilized cardiac extracellular matrix (cECM) derived from adult porcine left ventricle. Healthy porcine cECM mimics the native binding sites available to cells and has been shown to promote functional repair in a pre-clinical model of MI, but cECM alone is limited in terms of the ability to mechanically stabilize the infarct during remodeling as the gels formed by cECM are softer than normal myocardium (~3-7 kPa). This is important because hydrogels with a higher stiffness than native myocardium injected into the infarct have been shown to reduce pathological remodeling, indicating that modulation of stiffness may be an important tool to regulating healing response. The protein composition of the ECM also has a profound effect on cells and we, and others, have demonstrated significant changes to the cardiac ECM protein composition in normal development and in disease, which modulates cardiac cell proliferation, progenitor/stem cell differentiation, cell traction force against the ECM and paracrine signaling from stem cells in the infarct environment, among other effects. In particular, our lab has demonstrated the potential utility of ECM derived from younger developmental ages in promoting a more regenerative healing response, and preliminary data indicates that this also occurs in the context of adult injury as well. As younger animals have the capability of regenerating their hearts to a greater degree, a logical hypothesis is that this difference in regeneration potential is at least partially derived from the remodeling of the ECM in which the cells reside at that point in development. We propose the development and use of biomaterials that can dynamically change to mimic a more developmentally regenerative niche for cardiac tissue repair. Our central hypothesis is that injectable, highly elastic silk-cECM composite hydrogels containing fetal heart derived cECM will provide a means for mechanical stabilization of the infarcted ventricular wall while promoting more favorable remodeling by cardiac cells, leading to improved repair and the minimization of diastolic dysfunction. To assess this hypothesis, we will use combinations of silk-based crosslinked hydrogels and a series of relevant chemical modifications to achieve physiologically relevant mechanical properties with a range of dynamics. The samples will be characterized for their physicochemical properties and evaluated both in vitro and in vivo. We will then assess the impact of the incorporation of young developmental age cECM to our dynamic silk hydrogels on cardiac cells in vitro and cardiac remodeling in vivo. We hypothesize that optimized silk-cECM based formulations will enhance diastolic heart function and reduce ventricular wall thinning following injection 1-week post-MI. Weekly functional assessment (echo) and endpoint hemodynamic functional assessment (PV loops) will be used to monitor cardiac function over an 8-week repair timeline in rats.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Species-specific differences and the complexity of cellular interactions involved in human pain perception have created challenges relying on conventional preclinical rodent models or simple human pluripotent stem cell (hPSC)-derived nociceptor models for novel drug development. Pain signaling pathways comprise of multiple neuronal, glial, and immune cell types within the peripheral (PNS) and central nervous systems (CNS), but the contributions of these cells in pain sensation is still under investigation. Moreover, these cells and the circuits they form exhibit region-specific characteristics, with unique positional identities along the body axis, and require a three-dimensional (3D) microenvironment with component parts—including end-organ tissues— to establish appropriate connectivity, plasticity, excitability, and functionality. Recognizing these challenges, the goal of this project is to bridge the gap by developing advanced stem cell tools to facilitate the creation of comprehensive and region-specific models for studying afferent pain circuitry. Building upon established technologies for region-specific spinal cord differentiation within my lab, this project will pursue three main objectives. Objective 1 involves developing scalable and systematic methods to generate region-specific sensory neurons and glial subtypes. Objective 2 focuses on creating and characterizing region-specific somatosensory organoids that faithfully mimic the in vivo microenvironment. Objective 3 aims to integrate hPSC-derived pain circuits into two tissue-engineered models for women's health—breast cancer metastasis and endometriosis— to determine how region- and subtype- specificity impacts innervation in the context of health and disease. Altogether, we will produce a suite of region-specific cells, circuits, and tissues that will deepen our understanding of the unique pain networks along the body axis and expedite the development of model systems for high-throughput screening of targeted analgesics. Furthermore, as a new investigator with multidisciplinary interests, this New Innovator Award will advance technologies in our lab that can be applied beyond pain research to a broad range of applications in neurological diseases, tissue engineering, and regenerative medicine.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY (See instructions): Declines in cognitive and motor function are early harbingers of pending Alzheimer's Disease and related dementia (AD/ADRD). New technologies for characterizing mobility and cognition have made significant inroads within the younger, healthier population segments but have not permeated all demographics, especially among older adults. Additionally, current approaches for monitoring cognition and mobility are limited to periodic assessments in laboratory and clinical settings, which fail to capture the subtle gradation and continuous deterioration of these domains. To address this gap, we propose a multi-modal system of wearable sensors that will provide data to enable breakthroughs in statistical machine learning and health informatics, allowing for a nuanced, continuous assessment of mobility and cognition. Critically, our combined hardware/software system will address the unique challenges of an aging population including the need for a health monitoring system which is both comfortable and easy to use. In Aim 1, we will develop a system of wearable eutectogel sensor patches that are flexible and breathable, transparent, and inconspicuous. These individual sensor devices, designed for long-term wear, will provide direct, continuous, real-time monitoring of eight biophysical domains (gait, posture, head motion, heart rate variability, respiration, location, orientation, and movement). In Aim 2, building on state-of-the-art methods for the modeling and analysis of multiple time series, we will develop statistical machine learning algorithms and models for the estimation of cognitive function and mobility over time given data from the gel-biosensors. To validate the resulting engineered system, we undertake a feasibility study to determine the potential of the analytics-enhanced, integrated biosensor system and machine learning algorithms to predict cognitive function and mobility in 20 older men and women, starting in a controlled laboratory setting and extending into real-world scenarios. We anticipate our gel-based biosensor system and machine learning models will advance personalized sensing and health informatics with long term, direct application to detecting and predicting early development and progression of AD/ADRD that increasing numbers of older adults are now facing.

NIH Research Projects · FY 2026 · 2024-06

Project Summary Epilepsy is a debilitating neurological condition affecting 3.4 million Americans, with 0.6% of children dealing with active epilepsy. Mutations in the four main voltage-gated sodium channels active in the brain are leading causes of pediatric epilepsies, with mutations in SCN8A accounting for up to 1% of all epilepsy diagnoses. Many patients do not respond to current anti-epileptic drugs and current treatments focus only on treating seizures, but not on correcting the disorder. There is a critical need for new treatments for patients with SCN8A epilepsy. Exon 5 in SCN8A undergoes a highly conserved and developmentally regulated alternative splicing during development, with initially higher usage of the 5N “neonatal” exon, shifting gradually to predominant splicing of the 5A “adult” exon in late childhood. Recent studies show that Nav1.6 encoded by 5N-containing cDNA is less active than Nav1.6 encoded by 5A-containing cDNA in neuroblastoma cells, and that inclusion of exon 5A or 5N in SCN8A mRNAs can impact the effect of pathogenic mutations in other regions of the gene on Nav1.6 function. There are over 40 patients with SCN8A epilepsy that have mutations in exon 5 of SCN8A, that could potentially benefit from a splice-switching ASO therapy as it would prevent inclusion of the mutated exon, switching to the intact exon, without altering Nav1.6 protein expression. Further, the mechanism which regulates this switch in splicing from 5N to 5A in SCN8A remain poorly understood. We have optimized ASOs that induce a switch in splicing between exon 5N and 5A in both directions and developed a novel mouse model of SCN8A epilepsy with a pathogenic mutation in exon 5N. The ASOs we have developed can be used to study the role of each exon in brain function, neuronal development and susceptibility to seizures in healthy mice and as a therapeutic approach for patients with mutations in exon 5. We hypothesize that the alternative splicing of Scn8a exon 5 controls Nav1.6 activity and seizure susceptibility in the brain, and that ASO-driven splice switching to replace mutated exons with healthy exons in Scn8a mRNAs can reduce or prevent epilepsy, improve development and extend survival. First, we will evaluate the role of Scn8a exon 5 alternative splicing on brain activity, neuronal excitability, and seizure susceptibility. Second, we will determine the efficacy of an Scn8a splice-switching ASO in correcting Nav1.6 function and reducing seizure activity in a mutant exon 5N Scn8a mouse model. Third, we will identify RNA elements and splicing factors that regulate the splicing of Scn8a exons 5N/5A and use this knowledge to design new ASOs with distinct regulatory effects. These studies will shed light on the regulation of Nav1.6 activity, findings that could be applied to the other sodium channels in the brain, which are highly conserved and undergo a similar pattern of splicing regulation. The development of a disease-modifying treatment for patients with mutations in exon 5 of SCN8A may not only reduce seizures, but also aid development, increase functional skills, improve quality of life and reduce mortality in patients with severe DEE.

NIH Research Projects · FY 2025 · 2024-04

Abstract Lymphangioleiomyomatosis (LAM) is a genetic disorder caused by bi-allelic inactivating mutations in tumor suppressor genes TSC1 or TSC2, leading to progressive lung destruction and eventually the requirement for lung transplantation. Rapalogs, the only FDA-approved treatments for LAM, induce a cytostatic effect with stabilized lung function. However, lung function continues to decline upon treatment cessation. Thus, there is an urgent need for better treatment, potentially leading to a cure for this devastating disease. The monogenetic nature of LAM makes messenger RNA (mRNA) replacement an attractive therapeutic modality, which requires safe and efficacious delivery of functional mRNA to the lung and specifically into TSC- deficient cells. We have developed lung-specific synthetic lipid nanoparticles (LNPs) and showed in our preliminary data that in vivo organ-selectivity of LNPs can be precisely tuned by changing the linker structure in the lipidoid tails without complicating the LNPs formulation. Furthermore, we developed LAM cell-targeting hybrid LNPs that efficiently deliver functional TSC2 mRNA into TSC2-deficient cells and suppress mTORC1 pathway. mRNA replacement therapy significantly reduced tumor burdens in preclinical LAM models. Remarkably, mRNA therapy induced T cell tumor infiltration in the otherwise immune cold tumor microenvironment. We hypothesize: 1) improved lung-specific tumor-targeting LNPs will assist developing mRNA therapy for LAM, which provides a novel therapeutic strategy to achieve durable effects and complete response; 2) LNP-assisted mRNA therapy can reprogram LAM-like cells back to a “normal” state without affecting normal tissue, and can re-normalize LAM microenvironment; 3) multi-omic single cell analysis will reveal mechanism of action of mRNA therapy and help improve LNP design. Our hypotheses will be tested in the following Aims: Aim 1. To develop and optimize lung-specific synthetic lipid nanoparticles for LAM-targeted mRNA therapy. Aim 2. To determine therapeutic efficacy of LAM-targeting nanoparticle-assisted mRNA therapy for LAM. Aim 3. To determine mRNA therapy-induced LAM cell reprogramming and LAM microenvironment remodeling by integrative single cell multiomic analyses. The scientific and preclinical impact of this project are 1) development of LAM cell-targeted mRNA therapy; 2) proof-of-concept that mRNA therapy can achieve durable response; 3) molecular understanding of LAM cell reprogramming and LAM microenvironment restoration by mRNA therapy. The success of this study will open a new therapeutic paradigm of mRNA therapy with the potential of durable effects and complete remissions.

NIH Research Projects · FY 2025 · 2023-09

Project Summary 1) Objectives: We will define associations and pathways through which exposure to PM2.5 and metals contribute to dementia-associated neuropathology (DAN), incident dementia, and cognitive function. We will do so by leveraging resources from the Adult Changes in Thought (ACT) study, an ongoing, prospective cohort study of brain aging and dementia in older adults who are cognitively intact at enrollment. Every two years since 1994, ACT has collected vetted data on dementia, other brain health measures, physical health, lifestyle, medications, and residential history for over 5000 participants, following them until incident dementia or death. At each timepoint, ACT assesses dementia using the Cognitive Abilities Screening Instrument (CASI) and consensus diagnosis using DSM-IV criteria. AD is also assessed based on NINCDS criteria. For participants who consent to autopsy (~25%), ACT performs a neuropathological examination of their brains, including confirmation of dementia diagnoses. Our study will build on these data and resources to achieve 3 primary aims: (1) to characterize PM2.5 and metal concentrations within the olfactory bulb (OB), olfactory tract (OT), and brain tissues of ~140 human donors to establish whether the OB is a pathway through which air pollutants reach the brain; (2) to investigate the OB as a pathway for DAN within the OBs and brains of these donors; and (3) to assess the association of PM2.5, metals with incident dementia - including for pathologically-defined AD, μVBI, LBD, and mixed dementia – and cognitive function, controlling for key confounders and examining effect modification by sex, race/ethnicity, socio-economics, and health conditions and mediation by health conditions. 2) Approach: We will test our aims following a multi-disciplinary approach that relies on (1) our detailed analysis of brains and OB for particles, metals and DAN indicators for ACT participants who consented to autopsy and (2) our epidemiological analyses of the association of long-term ambient PM2.5 and metal exposures with incident dementia, AD, μVBI, and LBD for the entire ACT cohort and for the subset with neuropathology confirmed dementia diagnoses. For both, we will leverage ACT’s rich database of clinical and functional health measures, behaviors, and residential histories. We, for example, will use the residential histories to estimate long-term ambient PM2.5 and associated metal exposures for each ACT participant using novel spatio-temporal models. We will also use health and behavioral data to control for key confounders and predictors and health data to assess modification and mediation of the pollutant-dementia associations. 3) Expected Results: We will provide new evidence of the risks posed by airborne metals to incident dementia and of the pathways through which airborne metals cause damage. In so doing, results from our study will help identify targeted interventions to block pathways to dementia by type and mitigate the severe and growing burden of AD and other dementias.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Stress is a powerful biological force that fundamentally alters the brain and body and is a known important contributor to major adverse health outcomes like depression, anxiety and traumatic stress disorders, cardiovascular disease, diabetes, and overall mortality. While empirical research examining the brain and physiological pathways linking stress to the aforementioned major health conditions is nascent, the specific mechanism by which different types of stressors exert their influences across diverse populations remains largely underexplored. This gap is particularly significant for populations in which exposure to stressors, such as economic hardship and interpersonal aggression, is notably high. Such investigation is essential because it will allow us to better characterize mechanistic pathways linking experiences with economic and interpersonal stressors to health disparities and to devise potential strategies for addressing such disparities. Repeated exposures to these stressors likely trigger and amplify a cascade of stress-related brain and physiological responses that are known to mediate elevated risk for adverse health outcomes. In this project, we will examine several elements of this mechanistic cascade. We aim to apply validated scientific paradigms in novel ways to examine brain, physiological, and psychological responses to the recollection of specific personal experiences with interpersonal aggression, compared to other types of life experiences, among groups who are living below the median income, given that stress associated with interpersonal aggression is more commonly experienced among these populations relative to others. We will also use prospective smartphone-based ecological momentary assessment (EMA) methods to measure the frequency and severity of experiences with interpersonal aggression as they occur in daily life in real time, and we will associate these measures with brain, physiological, and health outcomes. We will examine the relationship between brain/physiological responses to interpersonal aggression and health outcomes and functioning measures – such as psychological distress, cardiovascular disease risk, cellular aging (telomere length), coping, emotion regulation, and social support – and determine whether brain and physiological responses mediate the relationship between extent of exposure to interpersonal aggression and health outcomes. We have assembled an interdisciplinary team with expertise in areas including neuroimaging, trauma, biomarkers of stress, physiological perspectives on health disparities, ecological momentary assessment, emotion regulation, and the empirical study of interpersonal aggression among populations who experience economic hardship, yielding a collaborative effort that is unique and synergistic. It is an approach with the potential to transform the way that neuroscientists and psychologists conceptualize and study stressors that occur at the interpersonal level, helping to overcome some of the obstacles that have prevented previous scientific investigation of personal experiences with interpersonal aggression. Among our central aims is to increase the understanding of the mechanisms underlying health disparities in order to inform the development of targeted prevention and intervention strategies for populations disproportionately affected by stress, consistent with NIH goals.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Alkylating agents in our environment from tobacco, pesticides, and produced during drinking water purification cause DNA lesions. These DNA lesions can cause replication fork stalling and lead to DNA double-strand breaks that are canonically repaired by the homologous recombination (HR) pathway. The RAD51 protein plays essential functions in the HR pathway and is regulated by proteins including the Shu complex (SWSAP1- SWS1-PDS5B-SPIDR), BRCA2, RAD52, and CSB. Misregulation of RAD51 regulators leads to genome instability and cancer. Recent studies from our lab and others identified novel additional roles of these proteins in non-canonical repair during DNA lesion recognition, response to replication stress, and transcription coupled repair of replication structures containing R-loops. Mechanistic insight from the yeast Shu complex determined a role during abasic lesion recognition and RAD51-mediated bypass mechanisms during replication. Our work shows that like the yeast Shu complex, the human Shu complex is sensitive to the prototype alkylating agent MMS and depletion of Shu complex components SWSAP1 and SWS1 cause reduced RAD51 foci. Whether the human Shu complex functions by a similar mechanism is unknown. Both RAD52 and Shu components SWSAP1 and SWS1 function during replication restart by an unknown mechanism. RAD52 may use its annealing functions during replication restart and R-loop resolution. The overall goal of this proposed research is to determine how the human Shu complex functions at stalled replication forks to recognize abasic lesions thus enabling either RAD51-dependent strand exchange or RAD52-dependent annealing repair activities. The experiments proposed in this research program will be conducted in two phases. During the mentored K99 phase, I will determine how the Shu complex functions to recognize alkylation-induced lesions like abasic lesions and modulate RAD51-dependent repair using training in cell biology, atomic force microscopy (AFM) and correlative optical tweezers-fluorescence microscopy (CTFM) techniques (Aim 1). During the mentored phase the candidate will take advantage of co-mentoring, resources available at the University of Pittsburgh and the UPMC Hillman Cancer Center for professional development to utilize these skills through research, mentoring, data presentation, and writing opportunities. During the independent R00 phase of the research program, technical skills obtained during the K99 phase will be applied to elucidate the role of Shu complex in RAD52-mediated replication fork restart (Aim 2). Also, during the R00 phase I will extend these approaches into a new area involving resolution of RNA-DNA hybrids by RAD52 protein complexes. These experiments will provide me with the data required for an early independent publication and preliminary data for R-series grants. Importantly, during the R00 phase the candidate will develop independence from their mentor and co-mentors by focusing on the dynamic interplay between the Shu complex and RAD52 in response to replication stress.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Non-communicable diseases (NCDs), including cancer, cardiovascular disease, diabetes, have become the greatest health threat for low and middle-income countries (LMIC). In Ghana, hypertension is recognized as a major public health challenge. The ubiquity of mobile phones in Ghana and the popularity of mobile communications make it possible to deliver mHealth interventions to our target population. However, there is crucial gap in knowledge of the effectiveness of mobile health technology-based interventions for hypertension management in SSA. The proposed AHOMKA mHealth platform is an evidence-based intervention to assist with management of hypertension in an urban and rural region of Ghana. AHOMKA is adapted from the Empower HealthTM system, a proprietary software application developed by Medtronic for direct patient-to- provider communication via a mobile application and text messaging for hypertension management. This US- Ghana collaborative R21/R33 research project will focus on adaptation of the Empower HealthTM system into the local context in an urban and rural region of Ghana by multi-level engagement with stakeholders. We have assembled a multi-national, multi-disciplinary team of researchers and medical device professionals with expertise in mobile technology development, hypertension, cardiology, population studies, and public health across multiple institutions in Ghana (University of Ghana, University of Health and Allied Sciences, Medtronic Labs Ghana) and the USA (Beth Israel Deaconess Medical Center, Tufts University). The proposed feasibility studies will be conducted at two large cardiovascular clinics in the Greater Accra region and the Volta region in Ghana. Phase One aims to (1) implement a user-centered, iterative design approach to adapt the mHealth platform based on feedback from stakeholders, and (2) conduct a 6-month usability and feasibility study with a cohort of patients. Phase Two aims to conduct a 12-month feasibility study to assess change in blood pressure among a cohort of patients in two regions of Ghana. In order to accelerate mobile health research in Ghana, we propose to form the AHOMKA Research and Education Network as part of the strategic capacity building plan.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Untargeted metabolomics using tandem mass spectrometry (MS) have attained substantial success in the discovery of biomarkers and advancing our understanding of cellular metabolism. Despite this success, only a small fraction of measured spectra can currently be annotated (assigned a chemical identity). This bottleneck can be attributed to the limitations of current annotation tools that have not yet exploited advances in deep learning and available data modalities (spectra, peaks, molecules, and fragments). The goal of this application is to advance the interpretation of spectra collected through untargeted metabolomics. We focus on annotating data collected through liquid or gas chromatology followed by MS, or MS/MS, as these three tandem technologies have become dominant technologies. Over the next five years, the plan is to harness deep learning to address three problems: 1) annotation, 2) translation between spectra measured under different instrument settings, and 3) explainable models for annotation, where explainability arises from connecting peaks to their respective molecular fragments. The Hassoun lab has extensive, relevant deep learning experience to effectively tackle these problems. The Lab also has experience in dealing with the nuances of metabolomics datasets. The Lab recently developed a novel deep learning annotation model that achieves 41% and 30% performance improvement over multi-layer neural networks and graph neural networks, respectively. Additionally, our lab has developed an ontology- traversal algorithm that yields correct-by-construction molecular substructures that can be assigned to peaks, thus giving rise to datasets that can be used to train explainable annotation models. The Significance of this research is that it addresses fundamental barriers that hinder developing deep learning annotation models. Our models and datasets will be released on GitHub to benefit biological and biomedical applications and metabolomics research. Because of their expected high accuracy and explainability, the models will expedite the interpretation of experiments, improve our understanding of cellular metabolism, and facilitate data sharing among labs. The innovation lies in maximally learn from data modalities and in creating models that exploit the learned representations. Further, the annotation and translation problems are formulated as a bidirectional mapping between domains, in contrast to current annotation models that assume unimodal mappings. These innovations are necessary to advance metabolomics research and they will open new research horizons in the field of metabolomics.

NIH Research Projects · FY 2026 · 2023-03

The Kritzer lab focuses on inhibiting protein-protein interactions involved in autophagy. Autophagy is a protein degradation pathway that is active in all human cells, and inhibiting autophagy shows promise as a therapy for late-stage cancers especially in combination with DNA-damaging chemotherapies. Autophagy research and drug development currently rely on compounds that inhibit autophagy indirectly. Better, more specific inhibitors of autophagy would be broadly adopted. A large amount of genetics and cell biology work supports that inhibiting the LC3/GABARAP family of proteins can block autophagy selectively. In one series of projects, we will develop novel stapled peptide and small molecule inhibitors of LC3/GABARAP and evaluate them in models of late-stage cancers and other diseases. Because of our expertise in compounds that bind LC3/GABARAP proteins, we also propose to evaluate related compounds as autophagy-targeting chimeras (AUTACs). These compounds could be used to selectively degrade any proteins in the cell, similar to proteolysis-targeting chimeras (PROTACs) but potentially more versatile and easier to develop. Based on strong preliminary data that validate the AUTAC concept, we will develop novel AUTACs and demonstrate their ability to degrade endogenous proteins, unlocking a broad new area for drug development in targeted protein degradation. Over the course of developing stapled peptides as autophagy modulators, the Kritzer lab encountered a common problem in the field: how to measure the amount that actually reaches the cytosol. In an independent series of projects, the Kritzer lab has developed novel assays that quantitate the cytosolic penetration of large-molecule therapeutics. In this proposal, we describe new opportunities to address challenging problems in drug development for large-molecule therapeutics. We describe new methods to measure penetration to different cellular compartments in any cell type, including primary cells. We also describe a molecular evolution approach to develop a new “turn-on” assay that measures the real-time kinetics of cytosolic penetration. We describe pooled CRISPR screens to reveal the cellular components that mediate endosomal escape. Finally, we describe a novel screening platform that will allow us to screen thousands to millions of molecules at a time for those that are most cell-penetrant; the new screen will be incorporated into a design-test-learn cycle to produce data-driven design algorithms for cytosol-penetrant molecules. All together, these data will represent a huge leap in our understanding of structure-penetration relationships for several classes of large-molecule therapeutics.

NIH Research Projects · FY 2026 · 2023-02

ABSTRACT Much of the world’s music has periodic rhythms with events repeating regularly in time, to which people clap, move, and sing. The ability to detect and predict periodic auditory rhythms is central to the positive effects of music-based therapies on a variety of neurological disorders, including improving phonological processing in dyslexia, enhancing language recovery after stroke, and normalizing gait in Parkinson’s disease. Yet the neural mechanisms underlying rhythm perception are not well understood, and progress is impeded by the lack of an animal model that allows precise measurement and manipulation of neural circuits during rhythm perception. Human neuroimaging studies indicate that perceiving periodic musical rhythms strongly engages the motor planning system, including premotor cortex and basal ganglia, even when the listener is not moving or preparing to move. Here, we test the hypothesis that the motor planning system is actively involved in learning to recognize temporal periodicity and communicates predictions about the timing of periodic events to the auditory system. We propose to take advantage of the well-described auditory-motor circuits in vocal learning songbirds and leverage the mechanistic studies possible in an animal model to test these ideas. Like humans (and unlike non- human primates), vocal learning birds have strong connections between motor planning regions and auditory regions due to their reliance on complex, learned vocal sequences for communication. Auditory-motor circuits in songbirds and humans have many structural and functional parallels. Recently, we showed that songbirds can readily learn to recognize a fundamental periodic pattern (isochrony, or equal timing between events) and can detect this pattern across a broad range of tempi. In Aim 1, we will test whether neural signals from premotor regions play a causal role in this ability to flexibly recognize periodic rhythms. In Aim 2, by recording in auditory cortex while reversibly silencing activity in a reciprocally connected premotor region, we will test whether premotor signals influence auditory processing of periodic rhythms. In Aim 3, by recording activity in a premotor region as birds learn to recognize isochrony as a global temporal pattern, we will determine whether premotor neurons develop sensitivity to temporal regularity and exhibit activity that predicts the timing of upcoming events. Establishing an animal model for rhythm perception will be transformative for music neuroscience, allowing detailed investigation of the neural mechanisms underlying rhythm perception and informing rhythm-based musical interventions to enhance function in normal and disease states.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY (See instructions): Traumatic stress can lead to alcohol misuse and alcohol use disorder (AUD). In particular, avoidance coping after stress (i.e., persistent mental and/or physical avoidance of stress-related stimuli) is associated with higher rates of alcohol misuse. Using an animal model, we have shown that exposure to predator odor stress produces persistent avoidance of predator odor-paired stimuli in a subset of rats, termed ‘Avoiders’. Importantly, Avoider rats show long-lasting increases in alcohol self-administration after stress, similar to findings in humans. The neurobiology underlying this phenomenon remains an open area of investigation. This K99/R00 award includes a comprehensive career development and research plan based on Dr. Marcus Weera’s preliminary data showing that Avoider rats exhibit increased tolerance to the aversive effects of alcohol, which is hypothesized to facilitate increased alcohol self-administration in these rats. Our preliminary data also show that Avoider rats exhibit blunted activation of lateral habenula (LHb)-projecting lateral hypothalamus (LHA) neurons by aversive doses of alcohol. The scientific goal of this K99/R00 award is to test the central hypothesis that LHA-LHb neurons mediate stress-induced tolerance to alcohol aversion and stress-induced escalation of alcohol self-administration in Avoider rats via three aims. In Aim 1, we predict that Avoider rats show blunted activation of LHA-LHb and LHb neurons in response to an aversive dose of alcohol, as measured by Fos immunohistochemistry and in vivo fiber photometry. In Aim 2, we predict that in vivo chemogenetic stimulation of LHA-LHb neurons rescues stress-induced blunting of LHb activity and stress-induced tolerance to alcohol aversion in Avoider rats, as measured by in vivo fiber photometry and alcohol conditioned place aversion, respectively. In Aim 3, we predict that in vivo chemogenetic stimulation of LHA-LHb neurons rescues stress-induced blunting of LHb activity and stress-induced escalation of alcohol responding in Avoider rats, as measured by in vivo fiber photometry and operant alcohol self-administration, respectively. Results from these studies will improve our understanding of the neural circuits underlying stress-induced changes in sensitivity to alcohol’s aversive effects and in alcohol self-administration. The career development goal of this K99/R00 award is to provide the principal investigator, Dr. Marcus Weera, with additional technical training and professional development, and to help him establish an independently-funded research program. During the K99 portion of the award, under the guidance of an expert team of mentors, Dr. Weera will expand his technical repertoire to include in vivo fiber photometry. He will also search for and secure a tenure-track faculty position. During the R00 portion of the award at his new institution, Dr. Weera will use his acquired skills to build upon these studies, gathering rigorous data for submission of an R01 application. The work and training supported by this award will be critical for the PI’s successful transition to an independent research career studying the neurobiology underlying individual differences in stress and alcohol responsiveness.