Harvard University

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT Patients whose lives have been permanently altered by limb loss are currently left with prosthetic options for restoration of function, but these are not perfect. Prostheses are limited in sensation, articulation, and integration, among other concerns. Complete restoration of the lost limb via therapeutic intervention remains a distant goal of regenerative medicine. This goal would be made significantly more tangible if a clear roadmap for how complex tetrapod limbs can be regenerated was elucidated. Such a roadmap would detail possible sources of progenitor cells and the cues that activate them to proliferate as well as instructions for how these substrate cells undergo the changes necessary for them to be competent to build a new limb and to do so with morphological precision. Since humans, mice, and other mammals do not naturally regenerate full limbs, the roadmap is not likely to arise from mammalian studies alone. However, salamanders, such as axolotls, are profoundly regenerative and can completely replace amputated limbs with precision throughout life, and, with experimentation, they thus offer a chance to build this roadmap. How processes that operate at the whole-body, systemic level to regulate progenitor cell activation and blastema formation remain critical unsolved issues in the limb regeneration field. A blastema is the bud-like structure that grows at the tip of the stump following amputation and contains the progenitor cells necessary to build a new limb. The blastema is essential for regeneration, and, except for the distal-most dip of digits, mammals do not form blastemas following limb amputation. Thus, understanding the controls, including the system-wide responses and inputs, that govern progenitor cell activation and blastema formation in salamanders is key to evaluating limitations observed in mammals. We hypothesize systemically-activated cells promote local limb regeneration in axolotl salamanders. We further hypothesize that axolotls use the hypothalamus-pituitary-adrenal (HPA) neuroendocrine system to stimulate systemic activation and that the central nervous system coordinates injury responses to orchestrate limb regeneration. This project uses modern tools, such as cell sorting and transplantation, paired scRNA-seq and ATAC-seq, and genome editing, to understand the biology of systemically-activated cells. This project will also use implanted flexible-mesh sensors and live neuronal activity recording for evaluating how the axolotl brain responds to limb amputation and how its activity changes as limbs regenerate. This work will lay the basis for understanding how the CNS may be required in the limb regeneration process. This project will provide important molecular and systems- level insights into how a highly-regenerative tetrapod responds to amputation and, ultimately, grows a new limb. It is therefore likely to have implications in regenerative medicine and evolutionary biology.

NIH Research Projects · FY 2026 · 2024-04

Project Summary / Abstract For the past thirty years, FlyBase has provided a centralized resource for Drosophila genetic and genomic data to enable a community of over 4000 research laboratories to further their research. Drosophila is one of the premier model organisms that provides cost-effective help in elucidating the etiology of human genetic diseases. FlyBase provides expert curation, controlled vocabularies (ontologies), and community access to a broad array of Drosophila research data and resources, including genome annotations, transcriptomics data, information about mutant and transgenic fly stocks, and much more. The value of this information extends to the study of human health and disease. Future progress in functional genomics in Drosophila depends on FlyBase to keep up-to-date with the literature. To achieve these goals, FlyBase works closely with other model organism databases (MODs) and the Alliance of Genome Resources (Alliance). Our resource development plan has three Aims. Aim 1. Improve curation and data utilization. We will: 1) improve the efficiency of curation by assessing the priority of the curated data for the research community and focusing on the highest priority data, and improve the technical curation process; 2) utilize the time freed up by increased efficiency to add new data types to FlyBase to match new directions in the field; and 3) help the community make the best use of the data in FlyBase by providing integrated summaries, including visual representations, and improved searching. Aim 2: Maintain and strengthen the FlyBase ecosystem. FlyBase synergizes with a number of other databases in order to enhance access to Drosophila data and tools by the research community. These efforts help maximize the utility of resources at all sites and distribute high-quality curated information to multiple websites, tools, etc. Our collaborators include the Alliance, the EBI Single Cell Expression Atlas, the Virtual Fly Brain project, the DRSC Functional Genomics Resources, and the Gene Ontology Consortium. Aim 3: Harmonization and transfer of FlyBase data and processes into the Alliance. FlyBase is a founding member of the Alliance whose goals are to harmonize model organism and human data and make it accessible to a variety of users following FAIR plus 5 principles. We will further integrate actively curated Drosophila data and develop processes and tools to import and export these data to and from the Alliance. This effort will have a major impact on multiple research and clinical communities, including the fly community, by accelerating the development of integrative analyses and approaches for fly models of human diseases.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT The incorporation of C–F bonds into small-molecule therapeutics can dramatically improve their pharmacokinetic properties, including bioavailability, lipophilicity, and metabolic stability. In accordance with the profound positive impacts of fluorine incorporation, approximately 20% of all pharmaceuticals contain fluorine. Aryl–CF3 linkages are a particularly common motif, present in approximately 15% of all fluorinated pharmaceuticals. Yet, a major drawback of current catalytic trifluoromethylation methods is that commonly employed CF3 sources can be very expensive, limiting the economic viability of trifluoromethylation reactions on large scales. Therefore, the development of an efficient trifluoromethylation protocol employing an inexpensive CF3 source would significantly improve the discovery and synthesis of new fluorinated pharmaceuticals. Trifluoroacetate (TFA), which is produced annually on multi-ton scale, could serve as the desired inexpensive CF3 source. However, the decarboxylation of TFA to generate reactive CF3 radicals (●CF3) is notoriously difficult, due to the strongly electron-withdrawing nature of the CF3 group. Recently, the Nocera group employed a silver(II) complex to achieve the first example of visible-light-induced TFA decarboxylation using a well-defined metal complex. However, this method’s use of stoichiometric silver(II), as well as the incompatibility of the reaction conditions with oxidatively sensitive substrates, limit the practicality of this advance. The objective of this proposal is the development of a practical, catalytic trifluoromethylation method. The stoichiometric, silver(II)-mediated method developed in the Nocera group will be modified to be catalytic in silver, with the catalyst turned over electrochemically. This method will be applied to the production of other difficult-to- generate radicals that are relevant to the synthesis of pharmaceuticals. Additionally, a rational ligand design campaign will be undertaken to develop silver(II) complexes that are compatible with oxidatively sensitive functional groups. Finally, the catalyst ligand environment will be modified to stabilize ●CF3 in the secondary coordination sphere, allowing for catalyst control over the precise delivery of ●CF3 to substrates. The proposed research is expected to constitute a significant synthetic advance in the field of radical trifluoromethylation. The Nocera laboratory at Harvard University is the ideal environment to accomplish the proposed research and training goals in preparation for a career in academia. The Nocera group will provide invaluable training in many areas of inorganic chemistry, particularly in the development of photo- and electrochemically driven redox reactions and in the deployment of mechanistic techniques, including transient absorption spectroscopy and photocrystallography. Moreover, Harvard will supply numerous opportunities for skill development in scientific education and mentoring through the Derek Bok Center for Teaching and Learning. These factors make Harvard the optimal institution to pursue ambitious research goals and prepare for a successful future academic career.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Bacteriophages, phages for short, are extremely abundant and diverse in the environment but also very under sampled and understudied. A major factor limiting the characterization of more phages is the lack of fast and efficient methods to do so. Alghough, techniques have been developed to find the receptor of a given phage on its host cell, no techniques to find phages dependent on a given receptor have been developed. However, this second method may be even more important. Such a technique would allow a user to select a bacterial membrane protein or structure of interest and preferentially select environmental phages that require it for infection. This could be useful for understanding the fluctuations of bacterial membrane structures across different ecological systems or for informing which environmental phages will make the best therapies. This proposal will develop a novel and generalizable assay for receptor-guided discovery of environmental phages using co-culture. The current standard technique includes a classic plaque assay where a single host strain of bacteria is grown in a lawn with an environmental sample of phages. Each phage infecting the host bacteria causes a clearing in the lawn called a plaque, and unique phages are not identifiable based on the appearance of the resulting plaques. The novel assay proposed here adds a new visual signal by co-culturing multiple strains of bacteria in the lawn. Each strain is a single gene knockout and tagged with a unique fluorescent maker. Phages with infection cycles independent of any of the proteins knocked out will lyse every strain on the plate and leave no fluorescent signal in the plaque. Phages of interest will lyse only a subset of the bacterial strains in the lawn, and the fluorescent signal corresponding to the required protein knockout will show through the plaque. In this way the novel assay differentiates plaques by the dependencies of the original infecting phage and allows for targeted isolation of phages of interest in just one experimental step. This project will be completed in a highly interdisciplinary environment where the experimental protocols, computational analysis, and hardware can all be developed with equal rigor. This assay will be developed first in Escherichia coli as a model system and then generalized into Pseudomonas aeruginosa to move towards clinical applications. Knockout proteins will be chosen first to test the limits of the assay and later to search for interactions between phages and bacterial virulence and antibiotic resistance factors. Taken together, this work will develop a novel assay for targeted phage isolation that can be applied to ecological studies, single phage therapies, and construction of phage cocktails.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Chemical genetic approaches are powerfully enabling for biological discovery and therapeutics development. The identification of mutant alleles that enhance or suppress the activity of chemical probes may not only validate on-target mechanism but also drive deeper understanding of a small molecule’s binding interactions, molecular mechanism of action, and downstream biological effects. Recent breakthroughs in genome editing technologies enable the systematic mutation of endogenous proteins at scale and directly in cells, opening new research paradigms for chemical genetic approaches. Exploiting these new technologies, our prior work helped pioneer the development of in situ CRISPR-mutational scanning approaches to systematically profile protein target(s) sequence-function relationships in their native cellular environment. When leveraged with chemical biology, these mutations in the target can be exploited as discovery tools to study small molecule mechanism of action and target biology, allowing us to uncover mechanisms of allosteric regulation, cell signaling, and cancer vulnerabilities. This chemical genomics platform allows us to unlock the serendipitous discoveries that both chemical probes and cutting-edge genetic screens can afford. To push the boundaries of chemical genomics, these approaches and their associated tools will need to be innovated, expanded, and tested in biologically meaningful systems and contexts that address key questions in the field. Hence, this proposal aims to significantly broaden the scope and utility of in situ CRISPR-mutational scanning in the context of highlighting its potential to transform many facets of chemical biology: to (1) interrogate drug-target structure- function in primary cells, (2) integrate massively parallel single-cell multi-omic readouts of mutant phenotypes, and (3) illuminate the function, mechanisms, and biology of protein disordered regions as well as (4) multi-subunit protein complexes. We focus our future studies on (1) androgen receptor, a critical cancer gene, where integrating high-throughput, direct readouts of transcription factor function will be transformative, and (2) components involved in targeted protein degradation, where holistically interrogating the ubiquitin-proteasome system at scale is essential for understanding the whole pathway. Across these aims, the biology and mechanisms of various protein complexes will be deeply explored by leveraging mutant alleles with cell, molecular, computational, and structural biology. Altogether, accomplishment of these objectives will advance research paradigms for chemical genomics and will further illuminate fundamental mechanisms of small molecule action and protein complex function.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY ASXL1 is one of the most mutated genes in hematological disorders. ASXL1 mutations occur in all types of myeloid malignancies, including 45% of chronic myelomonocytic leukemia cases where they are associated with poor prognosis; and ASXL1 is the third-most mutated gene in clonal hematopoiesis, a condition defined by aberrant expansion of hematopoietic stem cells (HSCs), which increases the risk of cardiovascular disease and other hematological disorders. Despite the prevalence and significance of ASXL1 mutations, it is poorly understood how they are mechanistically linked with these hematological disorders as very little is known about the protein itself. ASXL1 is a scaffolding component of the Polycomb-Repressive Deubiquitinase (PR-DUB) complex, which antagonizes Polycomb-mediated repression to regulate critical gene expression programs. Many ASXL1 mutations are frameshift or nonsense mutations in the final exon that result in truncated protein products. While early work focused on the effects of knocking out ASXL1, recent work has shown that truncated ASXL1 is actually present at higher levels than the wild-type protein. This stabilizes and hyperactivates PR-DUB, ultimately leading to epigenetic dysregulation. However, the molecular basis of how ASXL1 truncation increases its abundance and whether this drives its disease function remain unclear. In my preliminary studies, I discovered a degron in the last exon of ASXL1 that is necessary and sufficient for its degradation by the ubiquitin proteasome system. This proposal will test my hypothesis that the highly recurrent ASXL1-truncating mutations remove this degron to aberrantly stabilize the protein, leading to formation of hyperactive PR-DUB complexes, de-repression of Polycomb-target genes, and HSC expansion. To test this hypothesis, I will use a fluorescence-based cellular reporter of protein stability to further characterize this degron and perform a CRISPR screen to identify the E3 ubiquitin ligase that recognizes the degron. Then I will establish the relevance of this mechanism to the clonal expansion phenotype in primary HSCs by CRISPR mutagenesis paired with single-cell RNA sequencing (i.e., Perturb-seq). In the long term, this knowledge will advance our fundamental understanding of ASXL1’s role in primary hematopoiesis, how clinical mutations disrupt that process, and propel the development of new therapeutic strategies for a wide array of hematological disorders.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Although the early embryos of humans and other vertebrates have pluripotent cells that can differentiate into all cell types of the animal, these flexible cells are absent in adults. In contrast, many invertebrate animals maintain pluripotency beyond embryogenesis, and harbor adult pluripotent stem cells (aPSCs) that enable whole-body regeneration. The long-term goal of the PI’s research program is to obtain a mechanistic understanding of how pluripotent stem cells are made, retained, and regulated in vivo such that they can regenerate any missing cell type in an adult animal. Across 750 million years of animal evolution, cells operate on conserved principles, thus invertebrate species that maintain pluripotent stem cells in adult animals can serve as informative model systems. The overall objective in this application is to identify the molecular and cellular mechanisms that regulate aPSCs called “neoblasts” in the acoel Hofstenia miamia. Hofstenia can regenerate any missing cell type and is amenable to high-throughput functional studies of regeneration. The rationale for choosing a new model system over planarians, the more established system for studying neoblasts, is that Hofstenia produces manipulable embryos in large numbers, allowing the use of methods such as transgenesis, currently unavailable in planarians, to answer outstanding questions about neoblast biology. The experiments proposed here will combine lineage-tracing methods with functional genomics approaches to uncover and characterize critical regulatory control of pluripotent cells, asking three major questions: 1) Which genome-wide chromatin regulatory landscapes and transcriptional programs control the establishment and retention of pluripotent cells in embryos? 2) Which chromatin regulatory mechanisms and cellular dynamics enable neoblasts to achieve pluripotency during regeneration? 3) Which cellular mechanisms allow neoblast progeny to assemble into functional tissues? Isolation of the neoblast lineage followed by transcriptome and chromatin profiling in Hofstenia embryos, techniques that have been feasible in the applicants’ hands, will be utilized to identify chromatin landscapes and their regulators. Transgenesis, a technique recently developed in Hofstenia by the PI, will be used to specifically label neoblast subpopulations to trace their fates. Live imaging of differentiating neoblast progeny and functional studies of cell adhesion genes will be used to study how fate decisions are integrated with cell adhesion to enable assembly of newly regenerated tissues. In all research directions, RNA interference or CRISPR-Cas9 gene editing, two approaches that are feasible in this system, will be used to study gene function. This proposal is innovative in its use of a novel model system that has enabled new approaches, including the labeling and isolation of specific cell lineages, for studying long-standing questions about stem cell biology and regeneration. This project will reveal basic molecular and cellular principles for the regulation of pluripotent cells that have the potential to inform the development of new applications in human regenerative medicine.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY The goal of the proposed research is to discover how variation in the number of corticospinal neurons (CSNs) causes variation in motor circuit function and dexterous behavior, thus linking cells, circuits, and behavior in the same system. The evolution of advanced cognitive and motor function is associated with cerebral cortex expansion, including expansion of CSN neuron number, and it has long been hypothesized that the expansion of CSNs in primates underlies their exquisite hand dexterity, such as that required for tool use. In support of this, lesioning studies have demonstrated a role for motor cortex, and CSNs in particular, in dexterous behaviors. However, CSN number has not been causally linked to dexterity, nor do we understand the basic principles of how expanding a cell population alters circuit activity and thus behavior. The experiments proposed here will test the hypothesis that animals with more CSNs have greater dexterity, thus synthesizing cellular, circuit-level, and behavioral discovery, thus achieving the Goal 7 of the BRAIN Initiative. Specifically, this research will compare subspecies of deer mice (Peromyscus maniculatus) that evolved in different habitats and have innate differences in dexterity as well as a difference in CSN number. First, it will use single-nucleus RNA-sequencing to characterize which population(s) of CSNs differ in abundance between subspecies and to identify candidate developmental mechanisms underlying CSN population expansion. (Aim 1). At the circuit level, this research will determine whether neural activity during a dexterity task and neural architecture, as assessed with viral tracing, differs between subspecies (Aim 2). Finally, in the candidate’s independent research program (R00 phase), she will use the tools and datasets generated in Aims 1 and 2 to manipulate CSNs to establish causal links between CSN number, neural activity, and dexterity (Aim 3). This project will take advantage of recent technological advances in high throughput experiments (e.g., single nucleus sequencing) and neural manipulation (e.g., virally delivered gene editing) to work in a non-traditional model system, affording the unique ability to capitalize on naturally evolved behavioral differences to elucidate general principles of how cellular and neural variation mediate behavioral variation. This research is part of a comprehensive training plan that combines training in comparative behavior, developmental and systems neuroscience, and computational analysis of datasets in these areas, as well as training in communication and mentoring. This plan will be mentored by Dr. Hopi Hoekstra at Harvard University, an expert in deer mouse comparative behavior, and Dr. Adam Hantman at the University of North Carolina Chapel Hill, an expert in the neuroscience of motor systems. Additional mentorship will come from an Advisory Committee in single cell sequencing analysis, developmental neuroscience, and analysis of neural recording data. Together, this opportunity will provide excellent training for an independent research career in a unique, interdisciplinary niche at the interface of molecular neuroscience, behavior, and evolutionary biology.

NIH Research Projects · FY 2026 · 2024-04

Project Summary The structural integrity of the skin is dependent on appropriate cell attachment to the basement membrane, which forms the foundation of the epidermis and separates it from the dermis. Laminins are the major component of the basement membrane, where integrin receptors form adhesive contacts and connect the intracellular cytoskeleton to the extracellular matrix. Integrins are heterodimeric adhesion receptors that link cells to components of the extracellular matrix and to other cells, and they are essential for cell adhesion, migration, and multicellularity. Laminin binding integrins are fundamental in attaching epithelial cells to the basement membrane. In humans, a well conserved family of proteins, the tetraspanins, have been shown to bind and form signaling complexes with laminin-binding integrins. Tetraspanins are a class of four-pass transmembrane domain proteins with diverse biological roles involving modulation of the trafficking, membrane localization, and signaling of their various partners. The interaction between tetraspanins and integrins remains an exciting area of research as in vivo evidence suggests the interaction is necessary for normal integrin function. The tetraspanin CD151 binds all laminin binding integrins but associates most stably with the laminin-binding integrin α3β1 (ITGα3β1). Silencing mutations in either CD151 and ITGα3β1 both result in epidermolysis bullosa (EB), a disease phenotype consistent with impaired laminin binding and basement membrane attachment. However, why integrin function is diminished in this disease remains unclear, and whether CD151 affects ITGα3β1conformation or affects its activity through other signaling mechanisms is unresolved. Integrins characteristically exist in three conformations which each conferring different ligand affinity. CD151 may bind to and stabilize a specific ITGα3β1 conformation. This proposal will use structural approaches to determine if ITGα3β1 signaling is CD151 dependent due to conformation regulation. Additionally, signaling may be dependent on other downstream signaling mechanisms. I will use a proximity labeling time course approach to define the dynamic response to integrin activation and the influence of CD151 on ITGα3β1 signaling. These approaches will determine how CD151 modulates ITGα3β1, which can be applied to understand general tetraspanin and laminin binding integrin signaling biology. Although tetraspanins have many interaction partners and are implicated in various biological processes, the mechanism by which they engage and modulate their binding partners remains understudied. Specifically, understanding the CD151-ITGα3β1 signaling complex and its role in epidermal adhesion could be exploited therapeutically to strengthen epidermis attachment in EB and potentially other diseases of the skin.

NIH Research Projects · FY 2026 · 2024-02

Project Summary The human brain is composed of an exceptionally high diversity of cells and brain function is orchestrated by complex interactions of these different types of cells. The enormous cellular diversity and complex organization of the brain have so far hindered our understanding of the molecular and cellular mechanisms underlying brain function. To date, it is still unclear how many different types of cells are present in the human brain and how they are organized. We have previously developed a spatially resolved single-cell genomics method, Multiplexed Error Robust Fluorescent In Situ Hybridization (MERFISH), which allows in situ gene- expression profiling of individual cells and hence in situ identification and spatial mapping of transcriptionally distinct cell types in complex tissue, including the brain. However, the technical limitations of MERFISH still prohibit a comprehensive spatial analysis of cell types and states across the entire human brain. In this project, we will extend MERFISH to enable higher imaging speed/throughput, 3D volumetric imaging, and epigenomic-transcriptomic multimodal imaging, and we will use these approaches to identify cell types and cell states, map their spatial organization, and predict cell-type-specific cell-cell interactions in the human brain, including the aging brain. In Aim 1, we will develop methods to drastically increase MERFISH imaging speed/throughput (by 10-fold or more) and to enable 3D volumetric MERFISH imaging of thick tissue samples, which will further increase the imaging throughput and greatly improve our ability to capture and map cell-cell interactions. We will also extend MERFISH to allow spatially resolved single-cell profiling of not only histone modifications but also DNA modifications, and combine transcriptomic and epigenomic MERFISH to enable spatial multi-omics. In Aim 2, we will use the above-described approaches to generate spatially resolved cell atlases and cell-cell interaction maps of several human brain regions, including several cortical regions (prefrontal cortex, primary motor cortex, primary visual cortex, and middle temporal gyrus), several regions in the basal ganglia (caudoputamen, globus pallidus, substantia nigra, and subthalamic nucleus), and the preoptic region of the hypothalamus. In Aim 3, we will use the MERFISH technology, including the newly developed capabilities described above, to study the human prefrontal cortex from donors of different ages, including advanced ages, to characterize how the composition, cell state, spatial organization, and cell-cell interactions of molecularly defined cell types in the prefrontal cortex change during aging. Our proposed studies will provide fundamental insights into the molecular and cellular architecture of the human brain and how it changes during aging.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Over the past several years, we have experienced the social and physical consequences of a viral pandemic; however, the cellular and molecular mechanisms of repair following such infections are not fully understood. The lung serves as the site of gas exchange. Due to this function, it is constantly exposed to noxious environmental stimuli—including bacteria and viruses. Following viral respiratory infection, many of the epithelial cells lining the airways are lost and must be reconstituted. The epithelial-intrinsic mechanisms underlying such recovery are well understood, but the role of the immune system in this process is not well defined. In close contact with epithelial cells in the distal lung are alveolar macrophages, innate immune cells capable of sensing diverse stimuli and integrating these signals into effector responses. Our previous work has shown that macrophages engage in reciprocal growth factor exchange with fibroblasts, a communication circuit we predict to be active in other cell types and contexts. In the lung, macrophages have been observed to help mediate inflammation and repair via communication with epithelial cells following injury, however, the role of these interactions in the context of viral infection remains unknown. Using a mouse-adapted influenza A virus, we observe a marked loss of epithelial cells followed by rapid recovery, all under a constant presence of macrophages. This study aims to determine the role of macrophages in this repair process. We hypothesize that optimal epithelial repair requires macrophages at specific time points and anatomical locations following viral-induced damage. We also propose that bidirectional crosstalk between macrophages and epithelial cells promotes tissue repair. The proposed work will serve to 1) define when and where macrophages are required for optimal repair following viral injury and 2) provide mechanistic understanding of macrophage-epithelial communication networks in the injured and regenerating lung. Completion of this project will allow us to identify molecular targets whose modulation may improve patient outcomes following viral infection. The Franklin lab is the ideal research environment to perform these studies. Dr. Franklin (sponsor) is an expert in macrophage biology and has previously unraveled interactions between macrophages and non-immune cells. Our location at Harvard Medical School also grants access to world-class experts and resources to complete our aims. For example, Dr. Carla Kim (co-sponsor) is an expert on lung damage and repair and will provide guidance on lung biology and epithelial progenitor cell function following damage. Together, my mentoring team and institutional resources will help me achieve the goals outlined in this proposal and allow me to grow both as a scientist and an individual.

NIH Research Projects · FY 2025 · 2024-01

Project Summary/Abstract Fibrosis is a hallmark of cancer that promotes proliferation, metastasis, and immune evasion by altering the tumor stroma which accounts for up to 90% of tumor mass. In fibrosis, pathogenic departure from homeostasis results in the excessive deposition of extracellular matrix (ECM) by myofibroblasts, creating discrete regions of non-resolving wound repair. These regions of fibrotic ECM become dominant regulators of cell phenotype, providing both biochemical (i.e. ECM composition and soluble factors) and biophysical (i.e. mechanical forces and material properties) cues to promote tumor progression and restrict immune cell infiltration. Soluble factors within the interstitial space of these fibrotic organs drain into surrounding lymph nodes (LNs) and induce fibrotic remodeling in the LN, a common sign of poor prognosis in cancer and other fibrotic pathologies. LNs have a distinct microenvironment known as the conduit system, which traffics antigens and serves as a migratory scaffold for lymphocytes. In health, its organization facilitates interactions between T cells and antigen presenting cells to ensure robust immune activation in response to cancer, pathogens, and injury. Fibroblastic reticular cells (FRCs) construct and ensheath this collagenous network and produce cytokines that promote T cell survival and homeostasis. Disruption of this ECM network leads to T cell dysregulation and depletion, implicating lymph node fibrosis in disease progression. The mechanisms of fibrotic initiation in the LN and the effect of LN fibrosis in T cell function is poorly understood, yet represent an attractive therapeutic opportunity. In other tissues, fibrotic remodeling mechanically stiffens the microenvironment, initiating integrin-mediated signaling cascades. I hypothesize that similar mechanisms drive LN fibrosis by FRCs, and seek to explore how remodeling of the LN microenvironment affects development of T cell responses in fibrosis and cancer. The first aim of this proposal (F99 phase) evaluates whether integrin signaling drives LN fibrosis by promoting FRC-to-myofibroblast differentiation. This will be accomplished in part by analyzing LNs from Idiopathic Pulmonary Fibrosis and melanoma patients with advanced mechanobiological (atomic force microscopy) and spatial-omic imaging (CODEX) methods to measure stiffness, ECM content, and FRC/T cell phenotypes. The knowledge and skills learned in Aim 1 are then applied in Aim 2 (K00 phase) to study the impact of fibrosis in tumor draining LNs on anti-tumor T cell responses in murine models of melanoma.

NIH Research Projects · FY 2026 · 2024-01

Project Summary The regenerative capacity of stem cells diminishes as we age. In the case of hair follicle stem cells (HFSCs), this loss manifests as a sustained dormancy phase without regenerating new hair follicles. Although we have gained substantial insight into the molecular differences between young and old stem cells and their niches, a major roadblock in identifying key genes underlying age-related changes in mammals is the lengthy process of generating experimental genetic models (e.g., overexpression or knockout mouse lines) and then aging them—the combination of which delays experiments by years. As such, there is a significant gap in our understanding of the specific genes and pathways that drive age-related changes in HFSCs. Furthermore, although there is evidence suggesting that old stem cells can be rejuvenated, specific genes that can lead to this reversion are unknown. Addressing these knowledge gaps will substantially advance our understanding of stem cell aging and provide a starting point for developing therapies for age-related declines in stem cell functions. This proposal capitalizes on the expertise, tools, and techniques we have established to rapidly test multiple candidates to identify genes that drive or suppress age-related dormancy in HFSCs and delineate the cellular and molecular mechanisms underlying these age-related changes. Findings from us and others show that secreted factors from dermal fibroblasts play a key role in regulating the extended dormancy seen in old HFSCs. We have already nominated one such candidate gene, Gas6, which we have shown can reactivate dormant HFSCs when overexpressed. In Aim 1, we will focus on delineating the cellular and molecular mechanisms by which Gas6 drives HFSCs towards more youthful behavior. In Aim 2, we will aim to identify additional genes that regulate HFSC aging. To enable gene testing in old mice directly and to enhance our throughput, we have established a strategy for rapid, in vivo genetic manipulation directly in old wild-type mice by delivering targeted genetic cargo with adeno-associated viral (AAV) vectors. This strategy provides a practical route to manipulate a relatively large number of genes in vivo, which has previously not been practical in the context of mammalian aging. We will also use SHARE-seq, a powerful method we developed to assess both transcriptome and chromatin changes in the same single cell. We will combine these techniques to gain insight into the cellular and molecular mechanisms by which Gas6 and other genes function to enhance the function of old HFSCs. Collectively, these discoveries will contribute to the basic understanding of stem cell changes that occur with age, provide the research community with much-needed tools to accelerate aging research in vivo, and pinpoint potential genes that can be harnessed to revert or halt age-related defects in HFSCs and beyond.

NIH Research Projects · FY 2025 · 2023-12

Minoritized communities have the highest rates of violence exposure. However, those affected remain underdiagnosed and untreated for resulting trauma symptoms as severe as suicidal thoughts and behaviors (STBs) and as consequential as reactive aggression and violence perpetration. 1,2 Importantly, Black and minoritized youth are more likely to experience traumatic events and be diagnosed with disruptive behavior disorders,3-5 which perpetuates past criminal stereotypes that persist in the public psyche such as the Superpredator myth 6,7 and leads to further violence exposure through over-surveillance and incarceration.8,9 Rather than effective mental healthcare access, minoritized youth are being exposed and re-exposed to cycles of violence. The leading intervention for aggression and violence perpetration among youth in the US has been incarceration. 8 There persists a perverse logic that incapacitation, which removes “would-be offenders” from classrooms and neighborhoods, leads to safer communities; yet increased incarceration rates have not lowered crime rates nationwide as violent crimes have actually increased. 7,8 Related, the past 50 years of randomized control trials (RCTs) testing interventions for STBs revealed that efficacy has not improved, treatment effect sizes are small, and there is no difference in efficacy among interventions.12 In short, interventions aimed at suicide prevention and violence reduction have fallen short. A major factor for why these interventions don’t show widespread success may be that violence reduction efforts have largely been limited to the study of aggression and STBs as phenomena present in discrete groups, yet these behaviors often co-occur to yield more severe clinical presentations. Although critical for diagnosis and treatment planning, little is known about potential cognitive mechanisms that can help describe the function of self/other-directed violence. The current proposal is focused on systematically describing the function of self-directed suicidal and other-direct violent aggression using behavioral models of choice behavior and qualitative interviews. An understanding of the constraints and preferences of individuals directly impacts treatment engagement and effectiveness—clinical decision-making is therefore critical.14 Focus on people from minoritized communities and their developmental risk factors may help mitigate race-based stress disparities in healthcare and help identify systemic needs for social services that reduce barriers to accessing treatment.22-25 This study has three main objectives: (1) to test the link between violence exposure and decision making (2) to test the link between decision-making and self/other-directed violence among violence-exposed youth; (3) to conduct a detailed diagnostic assessment with qualitative interviews exploring beliefs about the function of STBs and violence perpetration among youth identified through algorithms applied to electronic health records.

NIH Research Projects · FY 2025 · 2023-09

The proposed project will demonstrate the feasibility of generating a complete synapse-level brain map (connectome) by developing a serial-section electron microscopy pipeline that could scale to a whole mouse brain. This work will image 10 cubic millimeters, itself an unprecedentedly large dataset that may exceed tens of petabytes. Yet the mouse brain is 50 times larger. Reaching this ambitious goal will require advances in whole-brain staining, imaging, image-processing, analysis, and dissemination tools. We will scale and test these tools by producing a connectome of the hippocampal formation, a critical brain region for memory and spatial navigation. Specifically, we will define our volume of interest via microCT scanning of a whole brain. Then we will cut it into semithin serial sections and image them with multibeam scanning electron microscopy and ion beam milling. This technique images a thin layer of tissue and then removes it to reveal the next layer until each section is fully imaged, minimizing distortions caused by previous ultra-thin sectioning approaches. The imaging data will be processed by an improved version of our state-of-the-art pipeline. After quality monitoring and image compression, our automated system will assemble the full volume from imaged slices and then label tissue elements: neurons, glia, blood vessels, myelin, cell bodies, and synapses. This reconstruction will then be proofread and registered to the Allen Institute brain atlas, allowing us to relate our data to other types of data. Our analysis will identify cell types by region and layer, and reveal the detailed connectivity of hippocampal formation circuits. Using custom software, we will integrate these structural results on cell types with other approaches based on light microscopy and single-cell gene expression, allowing us to relate our results to the extensive literature on hippocampal formation structure and function. To accelerate our research and dissemination activities, we will involve high school, undergraduate and graduate students in the proofreading and scientific discovery phases of our work, offering trainees mentoring as well as research experience. We will turn these data into a lasting resource for the scientific community and the public by scaling up free access via online sharing tools to allow any interested party to render, proofread, or otherwise analyze the cells and circuits in this volume. To illustrate how this resource can be combined with other discoveries, we will define cell types based on their morphology and connectivity, characterize the relationship between these assignments and transcriptomic-based classifications, and integrate this information with previous work. Finally, we will define local and long-range microcircuit motifs in our data and use it to identify circuit principles and mechanisms of memory and spatial cognition, by testing and improving models of the hippocampal formation. Throughout the project, we will monitor key performance parameters, such as imaging throughput of a single microscope, to evaluate the feasibility and cost of scaling up to a whole mouse brain connectome.

NIH Research Projects · FY 2025 · 2023-09

Abstract During transcription of many cellular and viral mRNAs, RNA Polymerase II is inhibited by negative elongation factors and requires the positive elongation factor, pTEFb, to release this stalled state. The binding of the 7SK small nuclear RNA to the HEXIM adapter protein as a part of the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex normally keeps pTEFb sequestered within the nucleus and this interaction must be overcome for productive transcriptional elongation. The most well-studied example of the regulatory mechanisms that underlie transcriptional elongation is the HIV Tat system. Briefly, in order to release the stalled state of its transcript, HIV has evolved the viral Tat protein, which binds 7SK and displaces HEXIM to hijack pTEFb. On the other hand, some cellular factors, like BRD4, achieve the same outcome without displacing HEXIM. This proposal aims to gain a mechanistic understanding of the structural state presented to all transcriptional regulators encountering an HEXIM-driven inhibitory 7SK snRNP complex, and how capture of pTEFb is achieved by the specialized HIV Tat transcriptional factor. The aims will be: (#1) to understand if the basic mechanisms are maintained in HEXIM1 and 2 and if diverse positive transcriptional regulators capitalize on the mechanism, (#2) to determine the structures of the individual RNA domains present in 7SK- snRNA and (#3) to solve the structures of both HEXIM-bound and Tat-bound complexes.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Enantioenriched amines are prevalent functional groups found in various bioactive compounds and pharmaceuticals. Current methods for accessing them come with limitations, such as poor atom-economy, poor selectivity, poor reactivity, or requirement for high-pressure gases. Transition metal catalyzed cross-coupling reactions represent a powerful approach towards accessing enantioenriched amine compounds, which could overcome some of the inherent issues with current state-of-the-art. However, the discovery of new asymmetric transition metal catalyzed processes often requires exhaustive ligand screening and search for an optimal set of conditions that enable satisfactory activity, chemoselectivity, and stereoselectivity. In some processes, the best metal catalysts employed are those without any added ligands or with ligands to which no chiral equivalents exist, presenting challenging scenarios for enantioinduction. This proposal outlines an alternative approach towards the development of new transition metal catalyzed methodology, utilizing hydrogen-bond donor (HBD) catalysis to activate C(sp3)–O bonds towards a Ni-catalyzed cross-coupling reaction. This dual catalytic platform is a conceptually unexplored approach towards cross-coupling chemistry, wherein the role of the HBD is to accelerate the oxidative addition while simultaneously imparting stereoselectivity to the step through ion pairing. These roles have traditionally been assigned to ligand properties as the driver of reaction development, however this proposed research will demonstrate the feasibility of leveraging both catalytic modes to cooperatively engage mild electrophiles in an enantioconvergent C(sp3)–C(sp2) cross-coupling to generate amine compounds. Suzuki- Miyaura and Mizoroki-Heck type couplings of readily-accessible hemiaminal substrates will be explored by leveraging HBD-mediated substrate ionization for the generation of iminium ions. HBDs have been demonstrated to be effective catalysts for the generation of iminium ions, and their subsequent engagement in enantioselective trapping by standard nucleophiles. Unlike these traditional reaction profiles, the trapping of iminium ions by a metal is proposed here. Interestingly, various stereoselective Ni-catalyzed couplings of C(sp3)–O bonds have been reported to perform the best under “ligandless” conditions or with olefinic ligands that are not easily converted to chiral equivalents, thus highlighting the strategy of this approach. Successful execution of this proposal will reveal a new method for accessing enantioenriched amines and establish a proof-of-concept for the synergistic cooperation of HBD-catalysis with transition metal catalysis.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Suicide is a leading cause of death worldwide and the second leading cause of death for adolescents in the US. Despite advances in understanding of suicidal thoughts and behaviors and their treatment, the suicide rate has not decreased. One possible factor limiting progress in this area has been a lack of focus on understanding (and targeting) developmental mechanisms that may give rise to suicidal thoughts and behaviors. Interoception, the perception of internal bodily states, holds promise as a novel factor that may increase risk for suicide. Suicidal thoughts and behaviors center around inflicting harm to the body, yet surprisingly little work has tested whether and how disrupted interoceptive processes may contribute to risk for suicide. Given the centrality of the body in suicidal thoughts and behaviors and the unique interoceptive learning and processing that may occur during adolescence, altered interoceptive processing represents a novel risk factor that may advance understanding of suicidal thoughts and behaviors among adolescents. This proposal tests the associations between interoceptive processes and the occurrence of suicidal thoughts and behaviors. Eighty adolescents with and without suicidal thoughts and behaviors will undergo assessments of suicidal thoughts and behaviors, psychophysiological measurement of interoceptive processes, exposure to childhood trauma, and ecological momentary assessments of suicidal thoughts and behaviors and appraisals of body sensations. Aim 1 of this proposal is to test the association between perceptual interoceptive processes (i.e., interoceptive accuracy, sensitivity, and attention) and suicidal thoughts and behaviors. Aim 2 of this proposal is to test the association between appraisals and descriptions of body sensations and suicidal thoughts and behaviors. Aim 3 of this proposal is to test whether interoceptive processes are a potential mediator of the associations between childhood trauma and suicidal thoughts and behaviors. The proposed study’s greatest potential impacts are to use innovative strategies to study a novel potential risk factor for suicidal thoughts and behaviors, identify putative pathways that have clear translational value, and to potentially identify novel targets for intervention to treat suicidal thoughts and behaviors in adolescents.

NIH Research Projects · FY 2025 · 2023-09

SUMMARY High-throughput connectomics is needed to generate the TB-, PB- and EB-scale wiring diagrams of mammalian brains, but is limited to the few research institutes (e.g., Janelia, Allen, Max Planck) with sufficient infrastructure. As resource-rich as these institutes are, none are able to do a whole brain at nanometer scale on their own. The failure to broaden participation to a larger community is an obstacle to scaling connectomics. We propose a new and more affordable imaging strategy that will allow many more teams to engage in connectomics. High-speed electron microscopes for connectomics – e.g., multibeam SEMs – are rare and prohibitively ex- pensive. More common single-beam SEMs have sufficiently high spatial resolution, but are prohibitively slow for connectomics. We plan to increase the speed of single-beam SEM systems to the speed of multibeam SEMs without substantially increasing cost. Our strategy adds artificial intelligence to SEM architecture to re- duce the number and dwell time of pixels that need to be imaged at high-resolution without adversely affecting “segmentability”. With new software and standard computer hardware, we can turn single-beam SEMs into intel- ligent, powerful devices at negligible cost. We demonstrated a proof-of-concept of a smart scanning system that we engineered into a single-beam SEM. The modified SEM acquires a low-resolution/low-dwell time image of a brain slice at high speed. It then uses ultrafast ML algorithms to extract most of the wiring from these images, while at the same time identifying in real time those salient pixels that should be rescanned to improve signal-to noise in the final wiring diagram. We have achieved >10-fold speedup in image acquisition, and plan to increase the rate significantly more. A significant scale-up in the rate of connectomics demands comparable improvements in image processing (stitching, alignment, and segmentation). We have built computationally more efficient methods for aligning and segmenting connectome datasets. We will integrate these methods into a cloud-based platform that will allow researchers without significant computational infrastructure or expertise to process connectomics datasets. All data products and capabilities will be publicly accessible through BossDB. In summary, this integrated research program will scale connectomics to a much larger neuroscience community.

NIH Research Projects · FY 2024 · 2023-08

Project Summary. Youths receiving mental healthcare often have multiple disorders, distinctive treatment- relevant personal and family characteristics, and problems that may shift during treatment. For these youths, modular psychotherapies, in which providers select from a menu of therapeutic elements to build personalized treatments, may be especially appropriate. One well-studied modular youth psychotherapy is Modular Approach to Therapy for Children with Anxiety, Depression, Trauma, or Conduct Problems (MATCH). In some trials with intensive, one-on-one expert support for element selection, MATCH significantly outperformed usual care and evidence-based standardized psychotherapies; but in trials with less intensive (more practically feasible) support, MATCH did not outperform usual care. This discrepancy highlights a key challenge of MATCH and other modular psychotherapies: selection of treatment elements. Because MATCH allows any number of sessions implemented in any order, a virtually unlimited array of treatment element sequences could be selected. This flexibility supports personalization but may also impact the effectiveness of MATCH and other modular psychotherapies, because it can be unclear which modules should be used when. As in other modular therapies, clinicians using MATCH are asked to use clinical judgment and are provided with decision- making guidance based on past literature and client weekly assessments. But research has shown that statistical decision-making models using archival data very often outperform clinician judgment. Currently, no modular youth psychotherapies employ data-driven statistical models to inform decision-making. This is understandable: no such models are currently available. The proposed study will be an initial step toward filling this gap; it will use statistical models of archival treatment data to provide decision-making guidance in modular youth psychotherapy, aimed at enhancing its efficiency and effectiveness. Per NIMH Priority 3.2, the proposed project will inform “tailoring existing interventions to optimize outcomes” by “reanalyzing… aggregated clinical trials” using “computational approaches… to facilitate clinical decision-making”. Specifically, the project will aggregate data from six MATCH trials (N=602, ages 6-15), to: model short-term between-person and within- person associations between use of each treatment element and subsequent symptom change at different stages of treatment (Aim 1); use statistical learning to identify moderators of these associations between elements and subsequent symptoms (Aim 2); and, in a holdout sample, test whether agreement between the model recommendations and youths’ treatment course predicts long-term treatment outcomes (Aim 3). To inform future development of clinical decision guidance tools (after the F31), clinicians will be interviewed about how model-based findings might be used in clinical practice (Exploratory Aim 4). Ultimately, consistent with NIMH priorities, this work may inform clinical decision making in youth psychotherapy in several data-driven ways, with the goal of eventually enhancing the efficiency and effectiveness of modular youth psychotherapies.

NIH Research Projects · FY 2024 · 2023-08

Project Summary: Natural products play essential biological roles in producing organisms and have been a historically important source of medicines. Microbial natural products research has focused largely on environmental organisms; natural products, especially the potential small molecule virulent factor produced by pathogenic microbes, are much less understood. Legionella infection causes Legionellosis, which can be present in its non-pneumonic form as Pontiac fever or acute pneumonic form as Legionnaires’ disease. The fatality rate of legionnaires disease is about 10% due to complications and about 25% for those infected in the healthcare facility. While how the protein effectors of Legionella affect the host have been intensively studied, identifying the secondary metabolites Legionella produce and the roles these natural products play in Legionella infection are understudied. The proposed work will identify and characterize the potential bioactive nature products from the human pathogen Legionella. Using a gene-targeting approach to identify a potential novel metalloenzyme that catalyzes unusual oxidative rearrangement to generate the N-nitroso product, one homolog conserved in over 170 Legionella pneumophila subspecies and another homolog conserved in Legionella drozanskii was discovered. Activity assay shows this homolog catalyzes similar reactions but utilizes different substrates. Bioinformatic analysis of the gene neighborhood identifies the biosynthetic gene cluster (BGC) encodes resistance enzyme and a prodrug activating enzyme, possibly indicating the biosynthesis of potential bioactive metabolites. Aim 1 will identify the natural product and bioactivity from the biosynthetic gene cluster from Legionella to uncover the potential virulent factor. Aim 2 will initiate the characterization and mechanistic study of two enzymes in the BGC involving the biosynthesis of the potential pharmacophore to facilitate the understanding and pave the way for the inhibitor design of this new class of pharmacophore-producing enzyme pair. Successful completion of these aims will identify the chemical structure of the potential bioactive natural products from the human pathogen Legionella. In addition, two key enzymes involving the potential pharmacophore biosynthesis will also be characterized. Identification of those natural products and their biosynthesis will possibly reveal new virulence pathways that can be targeted to combat Legionella infection. Ultimately, the overall workflow will be generalized to investigate other novel natural products or potential virulent factors from the human pathogen Legionella to fight the emerging infectious disease by expanding the pool of 1) new classes of cytotoxic drug candidates/virulent factors and 2) new inhibition targets from the biosynthetic pathway.

NIH Research Projects · FY 2024 · 2023-08

Transition-metal catalyzed cross-coupling transformations are indispensable to pharmaceutical development and medicinal chemistry, allowing access to innumerable bond connections in extremely complex molecular settings. Despite its power, continued reliance on transition-metal catalysis poses many challenges. For example, a most pressing challenge in pharmaceutical synthesis is the extreme cost associated with the noble metals that form these precious, high-powered catalysts (e.g., Ru, Rh, Pd, Pt, and Ir). Furthermore, transition- metals are treated with strict regulation due to their elemental toxicity, imparting significant cost in the upstream synthetic stages of delivering a drug to the marketplace. Finally, continued reliance on transition-metal catalysis is unsustainable, threatened by the rapid depletion of raw materials at known global deposits and consistent supply-chain disruptions. Thus, the development of alternative strategies for mild and general bond formation is necessary for continued productivity in pharmaceutical and medicinal chemistry. Organocatalysis is an attractive surrogate to traditional transition-metal catalysis, leveraging readily accessible, inexpensive, and practical small molecules as catalysts. The identification of an organocatalyst with the ability to mimic the behavior of a transition-metal catalyst forms an ideal approach; a priori, such a strategy would be “plug and play,” invoking only a change in catalyst identity while preserving the nature of cross-coupling partners traditionally utilized in cross-coupling chemistry. If successful, this approach would address each of the challenges previously enumerated. The goal of this proposal is to design, synthesize, and develop a series of “pseudometal” organocatalysts to facilitate a vast array of catalytic cross-coupling transformations. These catalysts are termed pseudometal to reflect their ability to mimic the classical bond-breaking and bond-forming behavior of transition-metal catalysts. Specifically, this research plan details the development of ortho-dithioquinones as pseudometal organocatalysts, guided by principles of rational design, structure-activity-relationships, computational modeling, and hypothesis- driven experimentation. Our preliminary computational results direct us to ortho-dithioquinones due to the neutral Gibbs free energies predicted for oxidative insertion of these scaffolds into several s-bond types. In this research, rigorous mechanistic and characterization studies will profile the key principles inherent to organocatalyst speciation and the associated elementary steps, featuring stoichiometric studies, linear free-energy relationship analyses, and catalytic intermediate characterization. Guided by a rich mechanistic understanding, we will examine the synthetic capabilities of these organocatalysts through a series of cross-coupling transformations, including examples of C–N, C–O, C–SF5, and N–CF3 bond formation. Overall, this research will establish a new paradigm for sustainable, accessible cross-coupling chemistry, and will significantly contribute to medical research, pharmaceutical development, and fundamental knowledge in organic synthesis.

NIH Research Projects · FY 2024 · 2023-08

Project summary. Inflammation underlies majority of human diseases including diabetes, atherosclerosis, and cancer. These diseases are responsible for majority of deaths and represent substantial global health burden. Macrophages and T-cells, subsets of immune cells, have emerged as key mediators of inflammation. The role of biochemical cues in shaping the transcriptional response of these cells have been investigated. However, accumulating evidence has shown that physical factors also tune their phenotype and effector functions. Recent two-dimensional studies have shown that mechanical confinement directs the nuclear translocation of transcription factors in macrophages. Another study found enhanced T-cell killing of cancer cells stiffened through cholesterol depletion. These studies have contributed to the field of mechano-immunology that seeks to understand how physical factors direct immune cell fate. Recent mechano-immunology findings have laid the groundwork for my proposal aimed at determining how biophysical cues shape macrophage and T-cell cell behavior. We have developed a three-dimensional culture that allows us to interrogate how biophysical cues regulate immune cell trafficking and macrophage-T- cell interaction in the tumor microenvironment. We have already identified that naïve macrophages are more efficient at trafficking to tumors than polarized macrophages. Furthermore, macrophages adopt different shapes depending on their activation state and their local microenvironment. Our preliminary results show that T-cells have longer-lived interactions with rounded macrophages, compared to elongated ones. This implicates macrophage shape, a biophysical property, in regulating its interaction with T-cells. We will extend these findings by elucidating the role of matrix viscoelasticity on immune cells behavior and performing a rigorous immunophenotyping of these cells. In addition, the proposal will implement machine learning algorithms to high resolution spatiotemporal information obtained from live confocal imaging. This will unlock the potential to identify heterogenous phenotypic states and quantify their evolution over time. Further, the proposal will integrate confocal live imaging with the single-cell RNA sequencing data. Such detailed, single cell analysis will identify genetic programs that are responsible for heterogenous morphometric states. The proposed research will be significant because it is expected to yield mechanistic insights that have broad translational impact for a myriad of diseases where inflammation is the underlying cause. These include Alzheimer’s, atherosclerosis, arthritis, diabetes, and cancer, which represent a growing global burden. The pathology of these diseases is orchestrated by macrophages and T-cells. Insight into the mechanobiology of macrophages, T-cells, and associated intracellular, transcriptional, and epigenetic modifications will deliver novel therapeutic options. Analysis of morphological heterogeneity using machine learning algorithms will provide a useful clinical and research tool to monitor disease progression.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract During development of the cerebral cortex, cortical projection neuron subtypes extend axons to innervate distinct targets located at great distances (103-105 cell body diameters) from their nucleus-containing somata. This precise navigation and circuit development is regulated by growth cones (GCs): subcellular compartments at tips of growing axons that rapidly integrate extracellular signals to control development of neural circuits, then mature into synapses. Since GCs respond rapidly to chemical cues, while hours/days are required to transport molecules between GCs and their parent somata, GCs highly likely function semi-autonomously. Prior work has also revealed that local protein synthesis and degradation pathways in GCs are required for responses to some directional cues. However, function of most GC proteins in axon guidance and circuit development have not been identified, and even less is known about subtype-specific roles of GC proteins. This proposal combines recently developed subtype-specific GC purification with ultra-low-input proteomics to 1) investigate subtype-specific GC proteomes at distinct developmental stages (pre- and post-midline crossing) to identify and functionally investigate proteins with stage-specific roles, and 2) investigate subtype-specific proteomes of dysfunctional GCs to identify and functionally investigate dysregulated proteins with critical roles in precise circuit wiring. These rigorous in vivo investigations will deepen understanding of subtype-, and stage-specific mechanisms regulating subcellular proteostasis in GCs, and how these processes control axon pathfinding and formation of synaptic circuitry in cortical projection neuron subtypes. The intersection of circuit formation and subcellular proteostasis has substantial

NIH Research Projects · FY 2026 · 2023-08

The ability to control and monitor the maturation of human-induced pluripotent stem cell (hiPSC)-derived tissues is critical for tissue engineering, regenerative medicine, pharmacology, and synthetic biology. This proposal presents an artificial intelligence (Al)-driven "cyborg tissue" platform that integrates tissue-like flexible electronic sensors and actuators with developing tissues and provides multimodal recording and control. Machine learning-based mathematical models will be built to integrate the data and tissue maturation status readout through the in situ single-cell RNA sequencing. This closed-loop system will control the tissue-wide distributed electrical actuations to promote tissue development. The aim is to use hiPSC-derived cardiac organoids as a model system to demonstrate that this Al-driven cyborg tissue platform can improve the maturation and eliminate the variations in patient-specific hiPSC-derived tissue samples. Specifically, flexible and stretchable mesh nanoelectronics with miniaturized multifunctional sensors and electrical stimulators will be fully implanted, integrated, and distributed across the entire three-dimensional (3D) volume of organoids for continuous, multiplexed sensing and actuation. Additionally, in situ electro-sequencing will be used to combine spatially resolved single-cell molecular phenotypes with the functional readouts from the electronics. A statistical learning architecture will be developed for modeling, testing, and interpreting multimodal electrical activities, mechanical contractile, gene regulatory, and signaling networks to determine the functional maturation of the organoids. Finally, a feedback control system will be implemented for real-time experimental design enhancement, electrical stimulation optimization, and model refinement to improve the functional maturation of cardiac organoids. The success of this work will potentially provide an improved mechanistic understanding of how genetic, molecular, electrical, and mechanical processes regulate the maturation of the hiPSC-derived cardiac organoids and establish an Al-controlled bioelectronics system to sense and control the functional maturation of hiPSC-derived cardiac organoids for various regenerative medicine and pharmacological applications. The technology is likely to be generalizable to help scientists understand the maturation and functions of virtually any kind of developing tissue and organoid systems and even in vivo systems. This proposed research will combine AI, machine learning, computational biology, biomedical informatics and multimodal cell data to advance stem cell maturation and enable new data-driven discovery, which aligns with the mission of the National Library of Medicine.