Harvard University

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Entry of nitrogen into the biosphere is crucial for the development and sustainability of life, as this element is utilized in the development of proteins, nucleic acids, and other cell constituents. Nature uses nitrogenase enzymes to mediate the transformation of the bioinactive atmospheric N2 into more reactive nitrogen sources such as NH3. Ubiquitous to all nitrogenase enzymes are multi-nuclear transition metal cofactors which act as the active site for N2 binding, reduction, and transformation into its reduced substrates. Despite the foundational role of the N2 fixation cycle to biology, the detailed mechanism of how nitrogenase enzyme metallocofactors facilitate N2 fixation is largely uncertain. Synthetic chemists are actively pursuing mechanistic elucidation of the N2 fixation by nitrogenase metallocofactors by using coordination chemistry to design molecular architectures which can bind and reduce N2. Significant progress has been made in this regard, especially in terms of the reduction of N2 with mononuclear transition metal compounds, but it is in question whether mononuclear systems provide accurate models for polynuclear metallocofactor sites. To this end, the use of polynuclear high-spin transition metal complexes as functional models for nitrogenase metallocofactors have been much less explored. The research program described herein involves the synthesis, characterization, and reactivity of new all monovalent [Fe3] molecular architectures which we propose could be functional models to explore the mechanism of the Fe- Mo cofactor (FeMoco) of nitrogenase. Initially, a new trianionic, hexadentate, ligand scaffold [NPL]3- will be synthesized which can support three monovalent first-row transition metal ions. The trimetallation of [NPL]3- with monovalent Fe(I) will be carried out to form the proposed monovalent tri-iron complex, (NPL)Fe3. Single-crystal X-ray diffraction, SQUID magnetometry, EPR spectroscopy, and cyclic voltammetry will be used to determine the structure, spin-state, and redox properties of (NPL)Fe3. The reactivity of the new [Fe3] cluster will be evaluated by reacting (NPL)Fe3 with simple chemical oxidants and nitrogenase substrates (i.e. N2 and CO). Compound (NPL)Fe3 will be subjected to reactions with oxidative N-group transfer reagents or N2 surrogates to establish the N-bound intermediates involved on the way to full N2 conversion. The oxidative group transfer reactivity of (NPL)Fe3 will also be explored using S-, O-, and C- group transfer reagents where the proposed sulfide complex, [(NPL)Fe3(µ3-S)]-, will be used to model the role of sulfide ligands in the N2 fixation of FeMoco. We will synthesize mono- and poly-hydride complexes, (NPL)Fe3H1-3, which could act as synthetic models for the E4 state of FeMoco, which is the intermediate proposed to bind N2. The reactivity of the hydride complexes will be explored with nitrogenase substrates such as N2, CO, and acetylene and the formation of the reduced amine of alkane substrates will be monitored. For promising model complexes, the efficacy of catalytic N2 or CO reduction will be investigated in the presence of a suitable chemical H+/e- sources or via electrolysis. If successful, these studies are predicted to elucidate key mechanistic steps of the N2 reduction process of FeMoco.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Aging is the largest risk factor for a majority of neurodegenerative disorders, including Alzheimer’s disease (AD) and related dementias. Despite growing incidence for such disorders, zero therapeutics are capable of reversing progression of aging-related cognitive decline seen in disease and healthy aging. Through the use of heterochronic parabiosis however, or the surgical joining of circulatory systems between young and old mice, our group demonstrated that blood-borne factors from young animals are capable of reversing many deleterious phenotypes seen in the aged brain. Understanding the molecular mechanisms underlying cognitive improvement post-parabiosis may uncover novel therapeutic avenues for aging-related neurodegeneration. One hallmark of aging linked to the onset of AD is the collapse of protein homeostasis (proteostasis) networks that regulate the folding of newly synthesized proteins via molecular chaperones as well as the degradation of pathologic misfolded proteins. During aging, cellular proteostatic capacity declines, leading to an increase in protein aggregates with physiological consequences driven by activation of stress response pathways in downstream cell types. Recently, by profiling transcriptional changes occurring in the aging mouse brain, our group identified chaperones that decrease in brain endothelial cells (BECs) with age, while simultaneously, in the same cells, levels of stress-inducible genes known to increase upon sensing misfolded proteins (e.g. Hspa1a, Hsp90aa1) were elevated – signatures which reversed post-parabiosis. Although BECs are some of the most vulnerable cells in AD, with many patients exhibiting altered blood-brain barrier (BBB) integrity, proteostasis machinery of these critical barrier cells has not been investigated. Taken together, in aging BECs, (1) molecular chaperones decrease, while (2) stress-inducible heat shock proteins (HSPs) increase, potentially reflecting the presence of misfolded proteins throughout the aged brain. To this end, we first seek to identify proteins that aggregate in the brain during aging and upon parabiosis via mass spectrometry. In doing so, we will profile aging-associated proteins which aggregate in a deleterious, yet reversible manner. Will then assess the ability of identified aggregation-prone proteins to activate stress response pathways in BECs. While our first aim asks, what is responsible for activation of stress-inducible HSP expression in aged BECs, our second asks what impact does increased HSP expression have on BEC function? This will be assessed by modulating Hspa1a (the most differentially expressed HSP in aging) levels using primary murine and human BECs alongside AD mutation-containing human induced pluripotent stem cell (iPSC)-derived BECs via lentiviral constructs. In parallel, we will modulate levels of Hspa1a in vivo specifically within BECs of mice via adeno-associated viral vectors. Expression of BEC markers and function will then be measured. The experiments outlined herein investigate roles for proteostasis and related stress response machinery in the aging brain, potentially identifying novel avenues for BEC/BBB-targeting AD and dementia therapeutics.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide exert their therapeutic effects by tailoring the substrate specificity of cereblon, a component of the ubiquitin proteasome system, resulting in altered substrate recruitment, ubiquitylation, and degradation. However, the native substrate specificity and biological functions of cereblon are elusive, despite the conservation of cereblon across species, its association with neurological development, and the potential impact that therapeutic engagement of cereblon would have on these substrates and pathways. To address the urgent need for better characterization of the native role cereblon plays in biological regulation, we undertook a novel approach to discover the substrate recognition mechanism of cereblon. We used a targeted protein degradation approach to systematically screen physiologically relevant ligands for functional engagement of cereblon in cells and discovered that we could replace thalidomide with peptides bearing a C-terminal glutarimide or aspartimide, which are post-translational modifications derived from cyclization of glutamine or asparagine, respectively. Endogenously, these C-terminal cyclic imides may act as molecular glues to recruit substrates to cereblon by tailoring substrate recognition, analogous to thalidomide, or may be generated directly on the substrate during protein aging and damage response or in response to signaling events. To address these two hypotheses, we will functionally characterize the recognition of these C-terminal cyclic imides by cereblon in cells in the context of two models: as peptide- based metabolites that alter substrate recognition or as part of a novel degron found at the C-terminus of proteins after protein aging or during a signaling event. We will first fully evaluate the recognition of peptide-based ligands in the context of bifunctional degraders for targeted protein degradation to characterize the scope and ligandability of cereblon by peptides and evaluate their ability to act as molecular glues for substrate recruitment in a manner analogous to thalidomide and lenalidomide. Next, we will assess whether C-terminal cyclic imides act as degrons that promote substrate recognition directly on engineered and endogenous proteins in biological systems. To facilitate the study of these modifications, we will develop orthogonal chemical labeling strategies to detect and map where and when these modifications occur in cells. Finally, we will investigate the formation of these modifications in cells to characterize the conditions, pathways, and “writers” that generate the C-terminal cyclic imides recognized by cereblon. The definition of C-terminal glutarimide and aspartimide modifications as the key recognition elements used to recruit endogenous substrates to cereblon and the associated studies constitutes a significant advance that will open new investigations into the biological regulation of proteins through these post-translational modifications and the effects on these pathways during cereblon engagement by small molecules, which will inform the use and development of therapeutics that engage cereblon in the clinic.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The developing mouse brain is a foundational experimental model for investigation of the origins of cell types in the mammalian brain. Comprehensive knowledge of mouse brain development is critical for comparative studies of neurodevelopmental processes, which are key to understanding the remarkable evolutionary innovations that distinguish humans from other species. In addition, developmental information enables refining cell taxonomy in the adult brain by incorporating knowledge of cell type and lineage origins into adult cell classification. Despite the transformative insights enabled by the recently created molecular atlas of the adult mouse brain, we currently lack a comprehensive census of cell types of the developing mouse brain, and the lineage relationships that link them to their adult counterparts. Here we seek to generate a comprehensive, spatially- and temporally-resolved, cellular-resolution atlas of the whole developing mouse brain, sampled at high resolution through the entire period of embryonic and postnatal brain development (from E8.0 to P28). We will employ three complementary approaches to generate comprehensive multi-omic single-cell profiles: 10x Genomics single-cell RNA-seq (scRNA-seq), 10x Genomics Multiome (simultaneous single-nucleus RNA-seq and ATAC-seq, for combined transcriptomic and epigenomic profiling), and Smart-seq3 (for full-length deep RNA-sequencing). In parallel, we will use the spatially resolved transcriptomic method MERFISH across the same densely-sampled timeline, to identify the spatial distribution of all cell types and dynamic changes in cell states across the entire mouse brain. We will apply computational methods to predict developmental lineage relationships from these spatially and temporally resolved datasets, and experimentally validate lineage relationships through both barcode-based in vivo lineage tracing and by functionally testing candidate molecular effectors using multiplexed in utero CRISPR screening (Perturb-seq). Finally, we will pilot integration of developmental datasets across species, mapping single-cell omics datasets from the developing human and non-human primate brains onto the comprehensive mouse brain developing cell type atlas established here, to create a computational alignment of developmental time that will enable understanding of differential regulation of specific developmental events across species. Overall, this project brings together a team of investigators with extensive, demonstrated expertise in brain development, circuitry, single-cell genomics, and assembly of brain atlases to produce a comprehensive developmental brain cell atlas, intended to serve as a first-of-its-kind foundational resource to the neuroscience community for the study of mechanisms of mammalian brain development and neurodevelopmental disorders. Our proposed project will contribute substantially to the overarching goal of BICAN to generate fundamental knowledge on diverse cell types and their three-dimensional organizational principles in the brain across lifespan and evolution.

NIH Research Projects · FY 2025 · 2022-08

Abstract In retroviruses, reverse transcription is initiated from an intermolecular duplex primer formed by nucleocapsid-driven annealing of the U5-primer binding site (U5-PBS) region of the genome with a host tRNA. However, the structure of this critical complex, and how reverse transcriptase (RT) interacts with it is largely unknown. The structure of the HIV-1 complex presented in this proposal shows that four tandem GNRA modules in U5-PBS spatially organizes the complex by making continuous tetraloop-receptor docking interactions: one engages a rearranged tRNA, one sequesters the initiation site, and two sequester the 18-bp primer to inhibit RT binding. Thus, in contrast to the widely accepted model that a single RT molecule recognizes this complex to initiate transcription, our data show that, in fact, two molecules of RT are required to bind in a step-wise manner to release the repressed state and ensure accurate initiation: the first extensively interacts with the U5 stem and acts as a remodeler, allowing for the subsequent one to bind the canonical 18-bp primer and perform the enzymatic activity. Manipulation of the architecture, the remodeling process, or competition with nucleocapsid, leads to severe loss of initiation accuracy. Thus, this study redefines our basic understanding of HIV reverse transcription initiation; assigns RT a structural remodeler role, separate from its enzymatic function; and indicates that the unique mechanism may contribute to the control of start of DNA synthesis in virions. The aims will be: (#1) to further detail the mechanism by mutational analysis, (#2) to understand the structural role of NC and (#3) to determine the structures of the remodeler RT and enzymatic RT bound to the U5-PBS:tRNAlys complex.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Diabetes mellitus is a complex disease associated with hyperglycemia. A growing body of epidemiological evidence supports that patients with comorbidity of diabetes mellitus and breast cancer are at a greater risk of poor prognosis and death compared with non-diabetic patients. However, the interplay between tumor progression and diabetes is still mechanistically unclear. Hyperglycemia results in glycation within the tumor extracellular matrix (ECM), where sugars crosslink collagen through a non-enzymatic reaction resulting in increased matrix stiffness. Notably, ECM stiffness is correlated with metastatic and promotes malignancy of tumors through multiple aspects, such as promoting proliferation and epithelial-mesenchymal transition (EMT). ECM stiffness also regulates endothelial cells, as our lab has shown previously that ECM stiffness promotes tumor angiogenesis and damages vascular integrity. Malformed and hyper-permeable vasculature is a hallmark of breast tumors and is known to lead to more metastasis and a more aggressive tumor phenotype. Noting the carcinogenic effect of glycation and the association between diabetes and tumor progression, my study aims to understand the mechanism by which diabetic hyperglycemia promotes breast tumor progression via glycation- mediated matrix stiffening. I have recently established a murine model where hyperglycemia was induced prior to tumorigenesis. With this model, I find that hyperglycemia increases tumor growth, tumor stiffness, advanced glycation end-product (AGEs) concentration, and EMT of tumor cells. Upon treating diabetic mice with glycation inhibitors, I observed a reduction of the previously tested metrics in diabetic tumors to levels comparable with non-diabetic tumors. These findings describe a novel mechanism by which diabetic hyperglycemia promotes breast tumor progression and provide evidence that glycation inhibition is a potential adjuvant therapy for diabetic cancer patients due to its key role of matrix stiffening in both diseases. In the F99 phase of this application, these findings will be extended by determining the mechanisms by which glycation-mediated ECM stiffening activates tumor angiogenesis (Aim 1). Noting that glycation stiffens ECM and produces AGEs which initiate cell signaling through RAGE receptors. AGE-RAGE signaling has been implicated in angiogenic behavior. A particular emphasis will thus be to tease apart the effect of AGE-RAGE signaling and matrix stiffening on tumor angiogenesis. Macrophage-involved chronic inflammation is emerging as a link between diabetes and breast cancer. My preliminary data show that there are more M2 macrophages within stiffer tumors. Thus, in the K00 phase (Aim 2), a research/training environment will be sought to examine the mechanism by how glycation- mediated ECM stiffening promotes tumor progression via increasing tumor immune cell infiltration and influencing immune cell behaviors. The ultimate goal of my studies will be to provide a more holistic understanding of how diabetic hyperglycemia influences tumor progression and to serve as a basis for future therapeutic intervention.

NIH Research Projects · FY 2024 · 2022-08

All organisms sense and adapt to changes in their environment. Alterations in organismal state can lead to epigenetic changes that dramatically influence how an animal interacts with its environment. How organisms acutely alter sensation based on their behavioral state is not well understood. Octopuses are incredible sensory specialists that use ‘taste by touch’ chemotactile sensation to interact with their environment. This sensory system is mediated by specialized chemotactile receptors (CRs) that detect poorly soluble molecules, such as those secreted by prey. If prey is unavailable for prolonged periods, do octopuses adapt to become more sensitive predators? One mechanism octopus might deploy to adapt is epigenetic adenosine to inosine (A-to-I) editing. Octopuses readily diversify their proteomes through editing mRNA transcripts by swapping adenosine for inosine, which is interpreted as a guanosine during translation. This process allows a single gene to produce multiple different translated proteins with potentially new functions. I will test the hypothesis that manipulation of organismal state biases preferential A-to-I editing to transiently alter protein sequence and function and modulate the detection of environmental signals most salient to the specific organismal state. Such a mechanism could tune the unique octopus chemotactile sensory system to be more sensitive to prey molecules when hungry. This project will utilize a multifaceted approach spanning from RNA biology to animal behavior, providing me with ample opportunity to learn new concepts, techniques, and establish an independent trajectory following my postdoctoral training. My diverse advisory team will provide expert guidance in cell physiology (Nicholas Bellono), RNA biology (Amy Lee), channel structure-function (Ryan Hibbs), and animal behavior (Venkatesh Murthy). In these studies, I will use molecular and biochemical approaches to identify which CRs are targets of RNA editing or are preferentially translated in response to distinct organismal states, such as starved versus fed (Aim 1). Our preliminary data demonstrate that octopuses edit protein-coding regions of CRs during periods of starvation. After identifying the spectrum of CR variants, I will characterize the biophysical properties of recoded CRs against unedited CRs to determine the functional consequences of state-dependent editing (Aim 2). I will focus my analysis on ligand sensitivity and ion permeation, which could account for increased sensitivity to prey molecules or altered neural signaling. Finally, I will leverage these discoveries to understand how the acute editing of individual proteins affects adaptive organismal sensation (Aim 3). I will carry out behavioral assays to test whether specific changes in protein function correlate with altered behavior across starved and fed octopuses. For example, if starvation-induced RNA editing of CRs alters sensitivity to prey molecules to enhance prey detection, I will test the threshold for chemically induced arm movement in behaving octopuses. Investigating how organisms can acutely regulate protein structure and function to alter behavior is novel and will provide fundamental insight into mechanisms of translation, signal transduction, and evolution.

NIH Research Projects · FY 2025 · 2022-08

Summary Animals actively sample sensory information, which they combine with prior knowledge to make decisions in a sensorimotor feedback loop. Aspects of this complex loop are often studied in isolation, using trial structures and in simplified conditions such as head-restrained animals in virtual reality. Studying an ethologically relevant, natural behavior in the laboratory can offer deeper insights about the behavioral strategies and their mechanistic neural implementation. Odor trail tracking is one such behavior, observed in many terrestrial animals including mice, and involves continuous re-orientation along the trail. The acquisition of odor cues is heavily guided by active sampling via sniffing and body movements, which introduces a strong coupling between sensation and motor actions. Theoretical studies hint at multi-modal strategies based on bilateral sampling, temporal integration and the use of internal models, whose relative contributions remain unclear. Here, a team of three PIs with complementary expertise, proposes to dissect the algorithmic and neural basis of olfactory trail tracking, which can offer deeper insights into active sensation, spatial navigation and continuous decision making. Using behavioral, physiological, molecular and analytical methods, the PIs will test algorithmic hypotheses and identify neural circuits guided by the following aims. In Aim 1, they will investigate the strategies exhibited by mice during trail tracking and identify brain regions supporting this behavior. A high-throughput adaptive system will be used to characterize the behavior of mice while tracking odor trails in a custom-built treadmill. In Aim 2, the PIs will uncover the neural circuits and cell types in brain regions involved in trail tracking. They will use cell-type targeted measurement of neural activity, viral tracing and transcriptomics in olfactory cortical areas to uncover patterns of activity and neural connectivity supporting neural computations necessary for trail tracking. In Aim 3, the PIs will elucidate, theoretically and computationally, behavioral strategies that mice use to track odor trails, and their underlying neural algorithms. They will use experimental data of Aim 1 to assess the validity of a novel theoretical framework, specifically in the context of sector search strategies and bilateral processing by rodents. Experimental data of Aim 2 will be used to unveil the neural dynamics and connectivity of sub-circuits that implement the algorithms driving behavior.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Individuals inherit one copy of each chromosome from each parent. However, the parental genomes within offspring are not functionally equivalent due to genomic imprinting, an epigenetic phenomenon in which certain genes are expressed from only one parental copy. Imprinted genes are widely expressed during development and play important roles in growth and neurodevelopment. Genomic imprinting increases disease susceptibility: genetic disruptions in the only expressed parental copy can result in imprinted disorders with frequent metabolic and neurodevelopmental symptoms. The Whipple lab seeks to determine (i) the molecular mechanisms regulating imprinted gene expression, including cell type-specific imprinted expression, and (ii) the cellular and physiological functions of imprinted genes, with a focus on imprinted non-coding RNAs. Fundamental discoveries related to genomic imprinting is expected to inform better treatments for imprinted disorders, as previously experienced in Angelman syndrome. (i) Using neuron differentiation as a model system to understand cell type-specific imprinted expression, the Whipple lab has recently identified parent-specific chromatin structure in the Kcnk9 imprinted domain that is strengthened during differentiation. This work will be extended to comprehensively quantify differences in the 3D folding of maternal and paternal alleles across imprinted domains. Future experiments will then probe the functional relationships between epigenetic modifications, chromatin structure, and gene expression in different tissues and cell types. (ii) Regarding the function of imprinted non-coding RNAs, the lab will primarily focus the next five years on discovering the targets and functions of imprinted small nucleolar RNAs (snoRNAs). snoRNAs typically guide chemical modifications on complementary RNA targets, but the targets of imprinted snoRNAs have largely evaded scientists for the past twenty years. Moreover, loss of paternally expressed snoRNAs are a major cause of Prader-Willi syndrome. The lab is developing new transcriptomic tools to discover snoRNA:target interactions, including an optimized snoRNA-RNA chimeric ligation approach for directly sequencing snoRNA targets. These findings will then be used to better understand the cellular and physiological effects of imprinted snoRNA activity and the pathways under parental influence in offspring. Through these efforts, the Whipple lab expects to uncover principles regarding epigenetic control of gene regulation and chromatin organization that broadly apply across diverse cell types, tissues, and organisms. The lab also expects to find new non-coding RNA functions with direct implications for understanding the biological processes dysregulated in imprinted disorders.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY / ABSTRACT The proposal outlines an integrated research and career development plan for Kayla Wolf, PhD, to complete postdoctoral training under the mentorship of sponsor Jennifer Lewis, ScD, and co-sponsor Lisa Satlin, MD. The overarching goal of the proposed project to develop a functional collecting duct network that is derived from human pluripotent stem cells and interconnected with a single drainage outlet. This network could serve as a model system for hypothesis testing, disease modeling, and drug screening or could interconnect directly with nephron-rich organoids to facilitate filtrate drainage in 3D kidney tissues. Recognizing that the extracellular matrix (ECM) plays a crucial role in development, this proposal with both investigate the role of ECM in collecting duct differentiation and leverage biomaterials as a powerful tool for guiding tissue form and function. Development of the collecting duct system will be accomplished by the completion of three aims. First, ureteric bud cells (UB, collecting duct precursors) generated from emerging differentiation protocols will be validated, and the effects of adapting the protocol to scalable culture methods on UB phenotype will be tested. Second, the role of extracellular matrix (ECM) composition on UB morphological and functional differentiation will be determined. Finally, the effects of fabricating a drainage outlet in a branching UB network on transport protein expression and function will be determined. Successful completion of this proposal will elucidate the role of ECM in driving collecting duct development, advance biofabrication methodology, and produce critically needed engineering solutions for generating functional kidney tissue. Dr. Wolf (PI) was supported by an NIH F31 fellowship (F31 CA228317-01) during her graduate research, where she established expertise in biomaterials, bioengineering, and mechanobiology. Under NIH F32 support, the PI receive extensive training in biofabrication and renal physiology at Harvard University, which cultivates a well- established, globally-leading biomedical research environment. The career development plan is designed to equip the PI with the necessary knowledge and skills for a successful career as an independent academic researcher.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY In the adaptive immune response, cytotoxic T lymphocyte (CTL) continuously “crawl” seeking evidence of foreign peptide fragments on the surface of other cells. Once the T cell encounters a target cell with foreign or mutant peptides, then it is activated unleashing a potent immune response. Emerging evidence suggests that cell mechanical forces transmitted to the T cell receptor (TCR) contribute to its high specificity in antigen recognition and promote T-cell activation. This is not surprising, as the TCR and other T cell co-receptors bind their cognate ligands only when two dynamic cells physically “touch”. As a first step toward understanding the role of molecular forces in tuning T cell response, it is important that we measure the magnitude of forces transmitted to ligand- receptor complexes and then to relate mechanical events to signaling and functional responses. My PhD research (F99 phase) has focused on developing methods to measure and elucidate the role of mechanical forces in immune response. I have designed a microparticle tension senor that allows one to quantify receptor forces in high throughput and also to measure forces at curved cell junctions. Additionally, I used this assay to screen the dose-response function of drugs that modulate cell mechanics. Because T cell responses are fine tuned by an array of co-receptors, I tested the role of mechanics in LFA-1 function. In this work, I demonstrated that the magnitude of LFA-1 integrin forces fine tunes TCR triggered activation and antigen discrimination. In addition, I revealed mechanically active LFA-1 defines the permissive zones for cytotoxic secretion, and suppression of LFA-1 forces significantly abrogates cytotoxicity. My work suggests that receptors cooperate to tune T-cell responses. For the remainder of my F99 phase, I will investigate the mechano-communication between receptor forces. Specifically, I will develop a DNA origami nano device to pattern ligands and measure spatiotemporal colocalization of mechanical events. Afterwards, I will proceed to test this hypothesis on cell plasma membrane by engineering tension probes on the surface of living cells. This will enable one to control and measure TCR-forces at authentic cell-cell junctions that mimic the chemical and physical properties of the immune synapse. For my postdoctoral work (K00 phase), I aim to improve upon current cancer therapies by leveraging T cell mechanics in boosting the specificity of immune response. In adoptive cell therapy (ACT), after therapeutic T-cell reinfusion, adjuvant drugs such as cytokines need to be administered to boost immune reconstitution. However, nonspecific drug release causes side effects and T-cell exhaustion. To address this challenge, I will decorate T-cells with DNA cages that mechanically trigger the release of encapsuled drugs at the tumor zone. If successful, this work will significantly enhance the ACT efficiency and offer the first example that links mechanobiology to cancer immunotherapy.

NIH Research Projects · FY 2025 · 2022-07

Summary: Over vertebrate evolution, the development of the myelin sheath has contributed to the expansion of the central nervous system and the emergence of complex brain function. Cumulative evidence indicates that the level of myelination and its positioning over the axon may be dependent on the class identity of myelinated neurons. A canonical example is the difference between L5 projection neurons, with extensive and uniform myelination, and the L2/3 callosal projection neurons, with lower and more diverse patterns of myelination, including “intermittent” profiles, where myelin tracts are separated by long unmyelinated regions rather than short nodes of Ranvier. Little is known about the mechanistic principles underlying cellular interaction between myelinating oligodendrocytes (OL) and axons of distinct neuronal classes in the CNS. Yet this knowledge is fundamental to understanding the cellular and developmental biology of myelination and regeneration. Focusing on the neocortex, we propose to answer fundamental questions regarding the mechanisms that control neuron-type specific myelination, and test hypotheses on how “attractive” and “repulsive” cues expressed by neuronal subtypes dynamically regulate their interactions with OLs. Here, we will 1) use molecular profiling of oligodendrocytes and cortical neuron subtypes across different cortical layers to map differences in their transcriptome, and use this data to generate a molecular interactome of candidates for genes mediating neuron- OL communication that may regulate neuron-subtype-specific myelination. We will 2) employ a screen to identify candidates able to induce or repress myelination (Aim 1). We will then 3) investigate membrane protein composition of myelinated and unmyelinated axonal segments of a specific neuronal class at subcellular resolution to understand the regulation of myelin positioning along the axon; and further 4) study whether long unmyelinated regions are differentially enriched for functionally-relevant structures such as synapses, gap junctions, and axonal branches (Aim 2). It has been reported that increased neuronal activity promotes myelination, which in turn stabilizes axon structure and neural circuit connectivity. Disrupted myelination can contribute to many debilitating neurological disorders, including multiple sclerosis and schizophrenia, and promoting oligodendrocyte differentiation and remyelination is an important therapeutic goal. We will investigate the molecular mechanisms that control cell-type specific adaptive remodeling of myelin and its regeneration after demyelination (Aim 3). In summary, the work proposed here aims to inform a conceptual framework for how different classes of neurons and oligodendrocytes interact to achieve differential myelination, mechanisms that will be critical in understanding the role of myelin in circuit function and dysfunction.

NIH Research Projects · FY 2025 · 2022-07

Project Summary This application requests support for the PhD Program in Chemical Biology at Harvard University. This profoundly interdisciplinary program trains highly talented students to carry out independent research at the forefront of chemical biology, addressing biological and medical problems using concepts and experimental approaches drawn from many areas of chemistry and biology and developing novel, broadly relevant technologies. The training program offers an extraordinary range of training opportunities in chemical biology. Faculty members at Harvard’s Cambridge campus offer world-class expertise in the disciplines spanning organic chemistry and the molecular life sciences, while faculty members at the Harvard Medical School (HMS) offer outstanding strengths at the interface between the molecular life sciences and biomedical problems. In addition, the Broad Institute of Harvard and MIT is a leader in technology development relevant to genomics, therapeutic discovery and medicine. The Program thus represents a mechanism to transfer concepts and technologies from chemistry to biology, medicine and genomics and vice versa. Students enter the program from a variety of backgrounds. Required coursework in the first year, provides core training in kinetics, chemical structure and reactivity as well as the application of thermodynamic concepts in the context of biology. Students are also required to take courses focused on reproducible research, statistics, and record-keeping as well as teamwork, communication and ethics. A unifying theme of first year coursework and discussions is the role of chemical tools and approaches in dissecting biological pathways. Students also take elective courses in synthetic organic chemistry, microbiology, cell biology, structural biology, genetics and genomics, proteomics, metabolomics and systems biology. They are exposed to an unusual range of technologies and concepts in chemical biology through coursework, interactions with faculty and other students, and Program events such as the Program retreat and Student Data Club. In addition, they develop communication skills, gain teaching experience and receive training in responsible conduct of research. Trainees are also supported in exploring a wide range of careers inside and outside academia. The previous grant supported 10 trainees and we request support for 10 trainees in this new proposal. The Program aims to attract a diverse community of talented students and give them intensive, personalized support as they develop an interdisciplinary understanding of biological questions and chemical concepts and approaches, and gain independence in posing and answering such questions. The program pays particular attention to rigor and reproducibility, training students in the physicochemical basis of the measurements they make and in appropriate techniques, and common pitfalls, in scientific analysis. Training for communication across disciplines and to the lay public is foundational for the Program and is emphasized throughout each student’s career. Communication is also foundational for multiple types of future careers.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY During embryonic development cells must be correctly allocated to distinct fates needed for organismal growth and reproduction. Germ cells generate eggs and sperm and must be specified to avoid disorders of the reproductive system, including gonadal and ovarian cancers, teratomas and other germ cell tumors, and ultimately infertility. Germ cells often acquire their fate by inheriting cytoplasmic components that are maternally synthesized, membrane-free, gel-like aggregates of proteins and RNAs collectively called germ plasm. The highly conserved proteins, RNAs and organelles within germ plasm are assembled, or nucleated, by other proteins that can be different in sequence across animals, but that share similar evolutionary histories and biophysical properties. The molecular mechanisms by which these nucleators ensure assembly and function of germ plasm remain unclear. Our long-term goal is to understand the molecular mechanisms that drive the assembly and function of cytoplasmic fate determinants. In this proposal, we will elucidate the mechanisms by which the Drosophila nucleator, encoded by the oskar gene, assembles germ plasm. This proposal is significant because it has the potential to uncover generalizable principles of germ plasm assembly, which may be broadly applicable to the formation and function of membrane-less, gel-like cytoplasmic aggregates that regulate cell fates in many different contexts. Our bioinformatic discovery of hundreds of new oskar sequences, combined with X-ray crystallography and biochemical assays, suggested previously unexplored hypotheses for the molecular mechanism of oskar function, which we will test as follows: In Aim 1, we will elucidate the role of the conserved LOTUS domain in germ plasm assembly with in vitro biochemical assays and in vivo transgenic assays of the biological function and biophysical properties of germ plasm, testing hypotheses regarding the importance of dimerization, higher-order aggregate formation, phase separation, and Vasa binding to germ plasm assembly. In Aim 2, we will determine for the first time the specific sequences and structural properties of the Long Oskar Domain that enable the Long Oskar isoform to recruit and anchor mitochondria in the germ plasm. In Aim 3, we will test the novel hypothesis that the conserved OSK domain interacts with specific classes of lipids in the oocyte posterior, to help anchor Oskar to the posterior pole. Since defects in germ cell development can lead to reproductive system pathologies affecting up to 12% of the US population, elucidating the mechanisms that ensure assembly and function of germ line determinants is highly relevant to human health. More generally, germ plasm is a type of ribonucleoprotein multimer (RNP), which are membrane-free, gel-like organelles that regulate translation in both germ line and somatic cells. Understanding germ plasm assembly may thus shed light on the general principles underlying assembly of cytoplasmic RNPs required for the proper function of multiple critical cell types.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Cancer cells, extracellular matrix (ECM) and carcinoma-associated fibroblasts (CAFs) are three critical factors contributing to tumor progression in colorectal cancer and squamous cell carcinoma, among other cancer types. CAFs are often associated with the development of high-grade malignancies of poor prognoses because CAFs secrete metastasis-promoting cytokines and abnormally deposit collagen, facilitating integrin- dependent cancer invasion and forming hindrance of anti-cancer drug delivery. Despite the detailed studies about the role of CAFs in tumor progression, the mechanism by which stromal cells become CAFs is still not clear: how normal fibroblasts in stroma are transformed to CAFs during early stages of cancer? In this project, I will test the hypothesis that cancer cells facilitate CAF induction by mechanically reorganizing extracellular matrix and thus breaking down the diffusion barrier of CAF-inducing factors. A coculture system containing colorectal cancer spheroids and multiple types of stromal cells as the testing platform will be constructed for the preliminary tests. If proven valid, I will use my hypothesis to develop new treatments where ECM remodeling is suppressed to decrease the rate of tumor progression in colorectal cancer. Two treatment strategies will be tested: (1) direct applying ECM crosslinking agents in the tumor microenvironment; (2) implanting genetically engineered fibroblasts secreting ECM crosslinking enzymes and other anti-cancer biomolecules. Colorectal cancer mouse models will be used to evaluate the efficacy of the new treatments.

NIH Research Projects · FY 2025 · 2022-03

Project Summary The family of monooxygenase enzymes are utilized to perform oxidative group transfer catalysis to broadly drive one of two functions: (1) metabolize hydrocarbon building blocks (e.g., steroids, fatty acids) for waste management or hormone synthesis in cytrochrome P450 (CYPs); and (2) the utilization of methane as the sole carbon and energy source (i.e., methanotrophic bacteria). The common trait amongst the oxidizing enzymes is the ability to electronically tune their catalytic centers to achieve oxygen transfer to robust C–H bond substrates. Adapting the electronic structure tuning principles to devise new synthetic, abiological catalysts holds great promise to (1) understand how the enzymatic systems might function by uncovering what reaction sequences are possible, and (2) developing new catalytic reactions that mimic the reactivity of the monooxygenases. This proposal describes the synthesis and characterization of novel metal-ligand multiple-bonds and metal-stabilized radicals to mimic the function of biological monooxygenases. Monooxygenases utilize metal- oxenoid ligands to drive C−H bond activation and C−heteroatom bond formation, providing a blueprint on how to emulate this reactivity. The ability to selectively incorporate functionality into unactivated C–H bonds represents a significant advance in converting inexpensive chemical feed stocks (e.g. hydrocarbons) to value-added functional molecules (e.g., pharmaceutical precursors). To achieve this goal, this proposal outlines a strategy to generate metal-ligand multiply-bonded complexes featuring oxenoid functionalities and examine their reaction chemistry as a function of transition metal and oxenoid ligand redox state. This proposal seeks to address the following questions: (1) Which transition metal-oxo linkage and attendant electronic structure can facilitate C-H bond hydroxylation chemistry? (2) Can monomeric copper support a terminal oxo-like ligand as would be suggested for the reactive oxidant in particulate methane monooxygenases? (4) How do functional group oxidation states (i.e., oxo, oxyl, oxene) impact functional group transfer catalysis? (5) Can metal-stabilized ligand radicals in general be developed to enable new C-H bond functionalization catalysis? Using dipyrrin ligand platforms as truncated models of the porphyrin platform found in cytochrome monooxygenases, this proposal outlines a strategy to synthesize and characterize metal-ligand multiple bonds on iron, cobalt, nickel, and copper. A sterically encumbered dipyrrin is proposed to be ideal for the synthesis, crystallization, and full spectroscopic characterization of a terminal oxenoid adducts of Cu akin to the potential terminal Cu(O) adduct in particulate methane monooxygenase. The broader scientific impact of the proposed research can be summarized as the following: this study will improve the field’s understanding of factors contributing to the promotion of productive C–H bond activation and functionalization, further developing new catalysts to synthesize value-added, commodity chemicals via clean reaction routes with minimal waste product.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY Building comprehensive accounts of human brain development from childhood to early adulthood is crucial to our understanding of both healthy neurodevelopment and the mechanisms underlying threats to youths’ mental health. Brain development unfolds on multiple levels, but the field lacks a comprehensive understanding of whether the trajectories of development are fundamentally similar or different across modalities (e.g., structure and function), and how they reflect developmental mechanisms associated with puberty or age (or both). The proposed research aims to generate a systematic and comprehensive multimodal account of typical neurodevelopment from ages 5 to 21, with a particular focus on a) identifying age versus pubertal- and hormonal-based mechanisms that undergird development in childhood and adolescence; b) systematic analyses across brain structure, function, and connectivity using state-of-the-art acquisition and analysis approaches; and c) evaluation of maturational variation in multimodal coupling across brain systems/ modalities as a function of development. Once established, this multimodal model of typical neurodevelopment will be used to test a conceptual model proposing that early pubertal timing leads to intensification of internalizing symptoms due to disruptions in brain development, including alterations in multimodal coupling. Specifically, models of the impact of early pubertal timing predict either further enhanced coupling with early puberty (neurodevelopmental acceleration) or disruptions in coupling due to neurodevelopmental delays. To compare these competing models and advance our understanding of the neurodevelopmental pathways by which early pubertal timing contributes to the rise of internalizing symptoms during adolescence, the research will use both hypothesis-driven methods and novel validated analysis pipelines for data-driven exploration of developmental changes in coupling, with careful attention to robustness and replication. The primary dataset will be the Human Connectome Project in Development (HCPD), a large, NIH funded, multimodal brain imaging dataset that includes a comprehensive assessment of brain structure, function, and connectivity paired with pubertal and hormonal measures and extensive behavioral and clinical measures in both a cross-sectional cohort of N=1300+ youth ages 5 to 21, and a longitudinal cohort (N=252) spanning ages 9 to 17 capturing the pubertal transition. This sample is purposefully strong diversity in race, ethnicity, and socioeconomic status. Key findings will be replicated to ensure robustness and generalizability in the Adolescent Brain and Cognitive Development (ABCD) longitudinal study. Our Specific Aims are to: A1) Establish a comprehensive, systematic account of age-and pubertal-linked pathways of brain development across multiple modalities of brain structure and function; A2) Test hypotheses about the relations of age and pubertal development to coupling across brain modalities; and A3) Test hypotheses about the neurodevelopmental mechanisms contributing to the rise in internalizing symptoms during the pubertal transition.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Skin stem cells are heavily influenced by signals from their niches including different fibroblasts populations. While our ability to isolate and molecularly profile diverse cell types has improved drastically in the past decade, a major roadblock in identifying key genes driving stem cell-niche interactions is the lengthy process of generating the genetic models needed (e.g., cell-type specific Cre or CreER, and overexpression or knockout mouse lines) to test gene functions in a cell-type specific manner in a physiologically relevant context. As such, while many different cell types have been identified and molecularly profiled, the critical genes that drive many developmental and regeneration processes remain incompletely understood. This substantial knowledge gap presents a significant impediment to developing therapies for skin diseases. To address this gap and to showcase how rapid functional genetics can enable new discoveries in stem cell- niche interactions, we will first build adeno-associated viral (AAV) toolkits to expand the field’s capacity for rapid functional genetics in multiple dermal cell types in mice. This aim expands on our current success in using AAVs to transduce dermal cells, with the goal of building tools that allows all skin researchers to modify gene expression rapidly in dermal populations such as the dermal fibroblasts and DP. We have recently developed and conducted SHARE-seq on the skin, a high-throughput single cell sequencing method that simultaneously measures chromatin accessibility (single cell ATACseq) and gene expression (single cell RNAseq) within the same cell. SHARE-seq data allow us to computationally infer key regulatory elements (enhancers, promoters) of signature genes for distinct cell types, which further enables the construction of cell-type specific AAV tools. We know the proposed strategy is feasible, because we have used it to build tools that can manipulate gene expression in the arrector pili muscles (APMs), a cell type that currently lacks specific Cre/CreER constructs. APMs are an emerging niche cell type for hair follicle stem cells (HFSCs). However, the molecular mechanisms by which APMs regulates HFSC behavior remain poorly understood. In Aim2, we will use our AAV tools to discover APM-derived secreted factors that regulate HFSC activation and maintenance. Collectively, these results will provide the skin community with much-needed tools to accelerate research in diverse topics, and may be relevant for understanding and potentially treating a wide range of alopecia conditions. Since AAVs are non-toxic and non-immunogenic, and since many key tissue-specific regulatory elements retain their specificity across species, there is an exciting potential to combine our biological findings with our technical advancements to develop novel gene therapy strategies to treat these skin diseases in the near future.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY/ABSTRACT In recent decades, the emergence of antibiotic resistance in bacteria has greatly outpaced the discovery of novel antibacterial agents. This research is focused on the synthesis and biological study of antibiotics effective against these modern pathogens of urgent threat. To this end, the lincosamides have been identified as an underexploited class of antibiotics. No new lincosamide has entered the market since clindamycin was approved more than 50 years ago (FDA, 1970). Growing resistance to clindamycin and its propensity to induce life- threatening Clostridioides difficile (C. difficile) colitis have limited its utility in today’s armamentarium. Due to the structural complexity of this class of natural products, semi-synthetic strategies are insufficient to support future antibiotic drug discovery within this or related scaffolds. Here, efficient synthetic pathways will be developed and implemented to prepare a large collection of lincosamide analogs inaccessible by any other means. These include analogs of a lead candidate, iboxamycin, which features a novel bicyclic oxepanoprolinamide scaffold and is efficacious in vitro and in vivo against a broad range of multi-drug resistant (MDR) bacteria. The latter include MDR ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), identified by the WHO as targets of highest priority in antibiotic development. By elucidating the mechanistic underpinnings and drivers of in vivo efficacy of iboxamycin and future lead antibiotics, this research will deliver multiple novel antibiotic scaffolds for preclinical exploration to target these challenging pathogens.

NIH Research Projects · FY 2026 · 2021-09

Abstract: The vision of the Generalist Repository Ecosystem Initiative (GREI) is to develop collaborative approaches for data management and sharing through inclusion of established generalist repositories in the NIH data ecosystem and better enable search and discovery of NIH funded data in the generalist repositories. This is one of the steps in the modernization of the data resources ecosystem and aligns with the NIH Strategic Plan for Data Science. The primary mission of the Generalist Repository Ecosystem Initiative is to establish a common set of cohesive and consistent capabilities, services, metrics, and social infrastructure across various generalist repositories. A secondary mission is to also raise general awareness and facilitate researchers to adopt FAIR principles to better share and reuse data. The objectives of the project are to work together with the other awardee teams to develop a “co-opetition plan” that include a commitment to a solution architecture, data submission and QA/QC capabilities to support relevant use cases, train and educate users, as well as provide usage analytics and reporting to the NIH.

NIH Research Projects · FY 2025 · 2021-09

Pancreatic islets rely on spatiotemporally orchestrated interactions between heterogenous cells to maintain blood glucose homeostasis. In type 1 diabetes, an islet-directed autoimmune attack leads to loss of functional β cells, which is accompanied by defects in the other islet cell types. Diabetics suffer complications from chronic glucose misregulation, which ultimately reduce life expectancy. Administering insulin itself can treat type 1 diabetes. However, daily insulin injection is expensive, onerous, and carries side effects including risk of ketoacidosis and coma. Human stem cell-derived islet organoids (SC-islets) offer a chance to generate a limitless human islet supply as potential therapeutics through transplantation. However, SC-islets lack the precision, kinetics, and magnitude of insulin/glucagon secretion that natural islets show during adult life. Whether these limitations reflect poor spatiotemporal coordination between (or within) populations of SC-islet cell types, or intrinsic three-dimensional (3D) heterogeneity in development and maturation, is still unknown. Here, we propose to address these fundamental questions by experimentally capturing the trajectories of cellular activity and interaction across the 3D volume of developing SC-islets through the integration of novel technologies from stem cell biology, soft thin-film nanoelectronics, tissue clearing and single-cell spatial transcriptomics, and computational and system biology. Specifically, we have (1) exploited scalable cell differentiation and purification methods to build “designer” SC-islets with custom α and β composition; (2) globally embedded soft stretchable sensor arrays within SC-islets, building “cyborg islets” for chronically-stable tracing of islet-wide α- and β-cell type specific electrical activities at single-cell resolution in vitro and in vivo; (3) implemented 3D tissue clearing, staining, imaging, and in situ single-cell RNA sequencing to spatially map hormones, biomarkers, gene expression, and cell types in the intact SC-islets at subcellular resolution; and (4) used fluorescently-labeled electronic barcodes to identify sensor positions within cleared SC-islets and computationally integrate chronic electrical recording with hormones, biomarker and gene expression data at the single-cell level. We propose to integrate and use these inventions to address major challenges in SC-islet maturation. Specifically, we aim to employ such multimodal characterization of SC-islet development to address (1) the role of Dec1 in islet maturation mediated by circadian entrainment; (2) the 3D heterogeneity in SC-islet maturation; and (3) the role of nerve innervation and vascularization in the maturation of transplanted SC-islets. The success of this proposal will result in a platform that can monitor the in situ single-cell activity of SC-islets in a chronically stable manner, provide an understanding of the 3D heterogeneity during SC-islet development and maturation. We envision that it will ultimately enable us to build functionally specialized and mature SC-islets for human therapeutics.

NIH Research Projects · FY 2025 · 2021-09

Genetic and neural mechanisms underlying emerging social behavior in zebrafish Our goal is to understand emerging collective behaviors of groups, such as schooling and shoaling in fish. Our approach is to dissect basic sensorimotor transformations in the zebrafish, which we believe play a fundamental role in explaining emerging social interactions. We have identified two simple and well described reflexive behaviors: 1) the optomotor reflex (OMR), where fish swim along with whole field motion stimuli and 2) object evoked re-orienting responses (OER) where fish turn away or towards moving objects, depending on the object’s size and movement. We have shown in preliminary modeling studies that an implementation of these two simple “motor primitives” in virtual agents can explain a significant fraction of the emerging social behaviors in adult fish. A compelling advantage of focusing our studies on these two simple reflexes is that they are robustly expressed in 7 day old larvae, which facilitates a detailed and quantitative behavioral analysis of the related visuomotor transformation, as well as a dissection of their underlying neural circuitry. A critical element in our proposal is the generation of mutant zebrafish that we have shown to display subtle but distinctive social behavioral phenotypes at the adult stage. We found that, even in the larval stage, and prior to onset of robust schooling and shoaling behaviors, these mutants already reveal behavioral phenotypes in the context of the OMR and OER, and that these phenotypical deviations are predictive of the later emerging differences in schooling and shoaling in adults. One of our central goals is the dissection of the specific changes in neural circuitry in the mutants that are responsible for these altered behavioral phenotypes. Some such changes in neural phenotype may manifest at the level of global brain structures, but many are likely to disrupt micro-circuits - either at the level of cellular identities or synaptic connectivity - that underlie both simple behavior in the embryo and more complex behaviors in the adult. Notably, we already have generated realistic circuit models that form specific hypotheses about the neural networks underlying the OMR and OER in wild-type animals, and these models are readily adjusted to identify and constrain the specific latent variables that are changed in the mutant animals. Such adjusted models serve as ideal priors and specific hypotheses to be tested in brain wide functional imaging experiments. Lastly, the identification of detailed neural phenotypes in mutant animals in terms of anatomical location, neuronal cell fate and synaptic specificity will facilitate linkage of these anatomical and physiological changes to specific cell fates and molecular pathways. Our parallel ongoing efforts in describing and modelling brain wide neural circuits in zebrafish (within the framework of the U19 Team-Research BRAIN Circuits program) will allow us to narrow down which of all these observable neural phenotypes in the mutants are responsible and causally related to the specific neural changes that underlie the changes in behavior.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Epigenetic clocks based on DNA cytosine methylation (DNAme) are currently the most robust biomarkers of aging in mammals. They can accurately estimate the age of diverse biosamples and are responsive to interventions–such as caloric restriction or rapamycin treatment—that are thought to slow aging. Despite their increasing ubiquity in the field, the biology underlying epigenetic clocks is poorly understood. Likewise, their potential as a readout to test pharmaceutical interventions and discover new aging-associated genes is yet unrealized. One of the main limitations to using DNAme clocks at scale is the cost of current methods. Commonly used technologies to assay epigenetic age, such as methylation chip and RRBS, measure hundreds of thousands of CpGs and cost hundreds of dollars per sample. An economical, targeted approach that can measure virtually any epigenetic clock is badly needed to enable larger experiments with epigenetic age as the primary readout. For the F99 phase of this proposal, I have developed a new method called Tagmentation-based Indexing for Methylation SEquencing (TIME-Seq) that enables targeted DNAme clock sequencing of dozens to hundreds of samples simultaneously. I have shown that TIME-Seq is capable of measuring diverse epigenetic clocks at a range of scales and decreases costs 1-2 orders of magnitude. I plan to validate the method via comparison to conventional methods and build novel epigenetic age predictors and biomarkers trained on age-related frailty metrics. In the K00 phase, I will investigate the biology driving epigenetic clocks using aging mouse muscle as a model. I will take a multiomic approach, using TIME-Seq and established genomic methods, to test the hypothesis that heritable loss of silencing at developmental loci, in part, drives ticking of the clocks. This proposal will provide the aging field with a much-needed method for cost-effective and high- throughput epigenetic clock assay, as well as novel biomarkers based on frailty, which can be used in academic research and a clinical context. Ultimately, the method will help us understand how and why it is possible to build DNAme clocks and what they tell us about epigenomic dysfunction during aging.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY Arithmetic and reading are the most fundamental skills acquired during elementary school and are crucial for successful academic achievement, employment prospects, and mental health. Despite the significance of arithmetical and reading skills, many children and adults in developed societies exhibit deficient reading and arithmetical abilities. Accumulating evidence suggests a strong neurocognitive and genetic link between reading and arithmetic, with high co-occurrence rates of both reading and arithmetical learning difficulties. However, the developmental trajectories of typical and atypical arithmetical and reading skills have been predominantly studied apart and potential shared mechanisms of typical and atypical reading and arithmetic development are unknown. This application proposes a longitudinal investigation that aims to (a) compare typical and atypical developmental trajectories of reading and arithmetic from kindergarten to third grade, (b) characterize similarities, differences, and classification power of neural and cognitive measures to profile the shared mechanisms underlying arithmetic and reading, and (c) determine a set of predictors in kindergarten predicting arithmetic and reading outcome after four years of formal instruction. Employing a longitudinal study design, the proposed project aims to investigate the developmental relationships of reading and arithmetic in the typical and atypical development of arithmetical skills from the beginning of kindergarten to third grade, in 180 children with either a familial risk for arithmetic difficulties (AD), a familial risk for reading difficulties (RD), or without any familial risk for reading or arithmetical difficulties, using functional and structural brain measures and behavioral correlates. Neural correlates of arithmetic, reading, and related sub- skills, together with a comprehensive psychometric battery measuring reading and arithmetical development and domain-general and domain-specific skills, will be examined four times over four years during critical stages of acquiring literacy and numerical concepts. By targeting children with a family history of AD and RD, and due to the genetic profile overlap of these two conditions, the probability of children exhibiting atypical reading and arithmetic development is increased, which will allow us to study and compare the developmental trajectories of both typical and atypical reading and arithmetical skills. This study has the potential to provide a model for understanding developmental learning disabilities, their underlying mechanisms, and their co-occurrence. The current focus on a reactive, deficit-driven instead of a preventive model in the field of learning disabilities is detrimental for students, as interventions have been proven to be most effective at an earlier age of heightened brain plasticity and because of the implications for mental health in struggling students as a result of a “wait-to-fail” approach. Understanding the shared and distinct underlying cognitive and neural mechanisms between typical and atypical arithmetic and reading skills and their precursors are of great significance for the development of early screening, diagnostic, and intervention tools.

NIH Research Projects · FY 2025 · 2021-08

Project Summary Natural selection and gene flow are important during species formation, and yet there is still much to be learned about how these forces shape the evolution of mutations that cause reproductive isolation. The overall vision for the proposed research program is to use functional genetic and genomic investigations to determine when and how selection and gene flow contributed to divergence during speciation. This research investigates reinforcement, which is the evolution of reproductive isolation in response to selection to decrease costly hybridization. Reinforcement can be a critical step during the speciation process, and yet there is little known about this process at the molecular level. The mutations causing reinforcement have not been identified for any organism, and the effect that reinforcement has on patterns of genetic variation throughout the genome has not been previously investigated. Three major goals of the proposed research are to 1. identify the mutations causing reinforcement; 2. infer the evolutionary history of the mutations underlying reinforcement; and 3. determine the extent of and gene flow during reinforcement. Mutations causing reinforcement will be identified using genetic association mapping near candidate genes and transgenic functional validation. Once the causal mutations are identified, population genetic analyses quantifying and describing variation surrounding the causal mutations will be used to infer how the mutations evolved through time. Specifically, this research will examine target genomic regions for evidence of selection, and determine if the causal mutations were from standing genetic variation or arose under selection. Finally, this research will use innovative comparative genomic analyses to quantify introgression across geographic space, throughout time, and along the genome of two species involved in the reinforcement. This research will be accomplished in the wildflower Phlox drummondii, arguably the best-studied case of reinforcement. Previous work provides a clear ecological and evolutionary understanding of how and why reinforcement occurred. Field experiments have confirmed that reinforcement caused divergence in flower color and two candidate genes causing this divergence have been identified. This system offers a unique opportunity to identify the mutations causing reinforcement and the signature of variation this process leaves across the genome. Understanding selection and gene flow during the evolution of functional genetic variants is critical to determining how all organisms, including humans, adapt to changing environments. This work is transformative because it develops a novel genomic framework for understanding selection and gene flow during reinforcement.