New York University

NIH Research Projects · FY 2025 · 2020-09

Project Summary Mechanical forces play a fundamental role in the behavior of many proteins within cells. Specific proteins have evolved to sense and alter their behavior in response to a stretching or compressive stress. This can happen in environments that are naturally under tension, such as cellular membranes, or in environments that are constantly rearranging due to active forces generated by molecular motors. In such environments, there is a close coupling between macroscopic forces at the level of entire cellular systems, and how those forces are sensed and generated by individual macromolecules. Predicting how these systems interoperate using computational techniques remains a challenge due to the need to span a wide range of length and time scales. Moreover, at the molecular level, the mechanical forces involved are quite small, and hence it is difficult to predict how proteins undergo significant changes to their conformational ensemble in response to these tiny perturbations. In our group, we have pioneered approaches to overcome this challenge, resulting in new computational methods that allow us to robustly predict both changes in conformational ensembles under tension as well as the force-dependence of unbinding rates using atomistic molecular dynamics simulations. We have also worked to make all our methodological advances available as open-source software tools. Currently, we are combining these new approaches to tackle mechanosensing problems of unprecedented complexity. We also work closely with experimental collaborators, including through joint mentees, and that work inspires our future to study load-bearing structures in both mammalian and bacterial systems. In this proposal, we describe planned efforts to extend our approaches to large biomolecular complexes and proteins in complex cellular environments such as bacterial membranes, which will necessitate further advances in simulation and machine learning approaches to treat these assemblies. Our studies will result in new and improved tools and techniques to share with the community, as well as insight into the functioning of crucial mechanosensitive motifs in living systems. Ultimately, our aim is to connect molecular responses to force to with emergent large scale mechanical properties of living systems.

NIH Research Projects · FY 2025 · 2020-08

How the brain and spinal cord transform neuronal activity into coordinated movement is a central question in neuroscience. Corticospinal neurons, the principle output of motor cortex, project to the spinal cord, where they shape motor output by synapsing on a diversity of spinal interneurons. Corticospinal neurons also send axon collaterals to the striatum, where they synapse on two populations of neurons with opponent roles in motor control. Intuitively, cortical projections the spinal cord and the striatum should be coordinated in the cell types they target, in order to direct coherent motor output. Experimentally addressing this possibility been restricted by an inability to map, measure, and manipulate interconnected circuits defined by their synaptic targets. Our narrow understanding of how these motor control circuits are coordinated has undoubtedly limited clinicians’ ability to treat movement disorders that originate in brain dysfunction, particularly Parkinson’s disease and dystonia. In this proposal, I will use recently developed technologies to uncover the anatomical and functional organization of cortical outputs to the basal ganglia and the spinal cord, and how these circuits are coordinated during movement. This proposal is organized in three Aims split across a K99 training phase and an R00 independent phase. In the first Aim, I will combine anatomical and electrophysiological tools to map the organization of synapses made by corticospinal neurons in both the spinal cord and the striatum. Experiments in this Aim will uncover a circuit through which these motor control structures are coordinated. In Aim 2, I will use two-photon imaging methods to define the logic by which corticospinal neurons, and their cellular targets in basal ganglia, encode salient features of movement. Finally, in Aim 3, I will combine anatomical, electrophysiological, and behavioral tools to determine how corticospinal neurons with collaterals in the striatum influence circuits in the spinal cord to shape motor output. Critically, experiments in all three Aims will capitalize on innovative technology developed and mastered at Columbia University, as well as interactions with expert collaborators. This work will be conducted in the newly developed and thriving Zuckerman Mind Brain Behavior Institute under the supervision of Drs. Rui Costa and Mark Churchland. Their technical and professional mentorship, along with that of expert collaborators in the Institute, will ensure I am fully equipped to lead a successful research program as an independent investigator. The experiments outlined in this proposal will become the foundation for my research program as an independent investigator and will have profound basic science and clinical implications for our understanding of motor control.

NIH Research Projects · FY 2024 · 2020-08

Project Summary It is well documented that a diverse workforce has the potential to reduce racial and ethnic disparities, which have strong effects in aging populations1-3. As the percentage of racial and ethnic minorities in the U.S. population increases, including among the aged population, the public health workforce should reflect this diversity1,4,5. With an increase in undergraduate public health majors across the country6,7, due in part to heightened excitement and opportunities in data science and quantitative big data analysis, and with breakthroughs in the science of aging on the horizon, this is an opportune moment to develop strong pipeline programs for underrepresented minority (URM) undergraduates. We will target URM undergraduates who have quantitative and computational interests and expose them to opportunities for graduate study and research careers, and the possibilities and excitement of marrying their quantitative interests with substantive research in aging. Introducing undergraduates, who are more diverse in public health majors than other majors7, and than graduate students6, to careers in public health, holds much promise for increasing the diversity of graduate students and faculty in the field, which has increased very little in the past 20 years6. This holds for the MSTEM subfields of public health, such as Biostatistics, Epidemiology and Data Science, as well. Motivated by these factors, and in response to NIA Funding Opportunity (PAR-17-290), “MSTEM: Advancing Diversity in Aging Research through Undergraduate Education (R25),” we propose an intensive, six-week summer program for 12 undergraduates from underrepresented backgrounds with interests in Biostatistics, Epidemiology, Data Science and other quantitative methods to learn about the applications of these methods in aging research. The summer program includes formal instruction, a broad lecture series, mentored research projects, oral research presentation at an annual symposium, career and professional development sessions, site visits to labs and other research settings, group and informal mentoring, social activities and network building, and training in responsible conduct of research. To reinforce the intensive summer experience, we will continue to offer group and individual mentoring and research experiences into the following academic years. Additionally, we will offer a select group of summer program participants the opportunity to return to NYU during the January term for an extended research experience and a quantitative course. With the guidance of a professional evaluator and internal and external advisory committees, we will evaluate all aspects of our program and review results in real-time to enable constant adjustment and improvement. Our proposal addresses three critical needs to strengthen and galvanize the research enterprise in the field of aging: increased engagement of MSTEM experts, increased engagement of URM researchers, and increased attention to disparities. We believe that our intensive and long-term programmatic components will support the entry of talented URM students into successful careers in MSTEM research in aging.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY Hearing is necessary for skilled acoustic behaviors like speech and music. While learning and performing behaviors like these, we listen to the sounds that our actions produce, we compare what we heard with what we expected to hear, and we detect mismatches between expectation and experience (i.e. errors) that can be used to update subsequent behaviors. The auditory cortex is critical for skilled acoustic behaviors like this, and auditory cortex cells integrate signals related to sound, behavior, expectation, and error. To understand how the auditory system works during behavior, it is imperative to learn how all of these signals are integrated within and used by hearing centers of the brain. However, it remains unknown how neural circuits integrate motor, acoustic, and goal-related signals to detect errors and guide learning. In this proposal, we will test the hypothesis that the mouse auditory cortex integrates sound and behavior-related signals during skilled acoustic behaviors, detects errors, and routes error-related signals to motor regions of the brain to adapt subsequent acoustic behaviors. To facilitate the use of lab mice for studying skilled acoustic behavior, we have developed a skilled auditory-guided forelimb behavior for mice that requires hearing. Using this paradigm, we will combine quantitative behavior with large-scale physiology, circuit perturbation, and anatomical tracing to determine how the auditory system detects acoustic errors and uses errors to guide skilled acoustic behavior. In Aim 1, we will first establish that skilled, sound-guided behaviors in mice require hearing and auditory cortex activity. We will then combine high-channel count physiology with behavior to identify populations of auditory cortex neurons that carry signals related to sound, behavior, and error. Finally, we will determine whether distinct auditory cortical error signals correlate with subsequent behavioral corrections. In Aim 2, we will first establish that motor cortex is necessary for skilled acoustic behavior. We will then combine electrophysiology with optogenetic “photo-tagging” to determine whether auditory cortex error signals are concentrated in neurons that project to the motor cortex. Finally, we will use optogenetic silencing to test whether auditory cortical neurons that project to motor cortex are necessary for driving behavioral changes. In Aim 3, we will first make physiological recordings from motor cortex to identify preparatory activity patterns that encode upcoming behaviors. We will then make simultaneous recordings from auditory and motor cortex to determine whether transient auditory error signals correlate with changes in motor planning activity on a trial-by-trial basis. Finally, we will ask whether auditory cortical error signals are necessary for updating motor cortex preparatory activity following behavioral errors.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY/ABSTRACT Changes in transcription factor (TF) binding in the genome and the regulatory output underlie many human diseases, including cancer and developmental disorders. Although extensive TF binding site (TFBS) maps exist, it remains challenging to predict how genome, epigenome, and cellular variation interact to shape genome-wide TF binding patterns and gene regulatory networks (GRNs). Major gaps remain in our understanding of how the context of TF-DNA interaction, including variation in TF protein sequences and DNA elements, chromatin accessibility, and cell identity, collectively influence these interactions and their downstream regulatory outcomes. The goal of this proposal is to elucidate how these layers of features jointly determine TF binding and reshape the GRNs that drive gene expression changes and cellular phenotypes. This proposed research leverages a powerful combination of advanced experimental techniques and computational modeling approaches developed and optimized in Arabidopsis thaliana, a genetically tractable model organism with extensive natural genome and epigenome variation and well-characterized cell types. Specifically, we will pursue three interrelated but independent projects, using the basic leucine zipper (bZIP) family TFs as a test case: (1) to develop integrative AI-based language models combining protein and DNA sequence information to predict how sequence variation in both TF proteins and their DNA binding sites modulates DNA binding of bZIP TFs in multiple species; (2) to use in vivo transient TF binding assays, coupled with parallel transcriptome and accessible chromatin profiling in Arabidopsis natural accessions, to systematically quantify how DNA sequence and native chromatin environments jointly regulate genome-wide bZIP binding and target gene expression; and (3) to map cell type-specific combinatorial regulation by systematically screening homo-, heterodimer and cross-family interactions for the Arabidopsis bZIPs, combining AlphaFold structural modeling, doubleDAP-seq TF-DNA binding assays, and single-cell CRISPR perturbation experiments to identify TF-regulated genes in a cell type-resolved manner. By addressing critical gaps in our understanding of the contextual determinants of TF-DNA interactions and transcriptional outcomes, the proposed studies will significantly advance our ability to model and predict context-dependent regulatory variation. The integrative modeling framework developed here will be broadly applicable, providing powerful tools to connect genome and epigenome variants with their molecular effects on TF activity and transcriptional output. These fundamental insights will have direct relevance for human health by enabling more accurate identification and mechanistic interpretation of regulatory mutations linked to complex diseases, thereby supporting precision medicine strategies targeting transcriptional regulation.

NIH Research Projects · FY 2024 · 2020-07

Map Leukemia-immune Cell Communication with Nanoplasmon Ruler in CAR T-Cell Immunotherapy Genetically engineered T-cells modified with chimeric antigen receptors (CAR) targeting CD19 provide an innovative method for treating cancer, especially for B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, practical application of this immunotherapy is greatly hindered by the unsatisfactory CAR T-cell function, long- lasting B cell aplasia and accompanied cytokine release syndrome (CRS). Improved therapeutic and preventive treatments require comprehensive understanding of the complex and dynamic cytokine secretion behavior of CAR T-cells and their communication with cancer cells and other immune cells in the tumor microenvironment. More importantly, real-time and traceable monitoring of both the location and timing of cytokine secretion would enable mechanistic understanding of CAR T-cell physiopathology in initiation, activation, communication and subsequent functional responses in leukemic bone marrow immunity. Such spatiotemporal monitoring technique is critically lacking within existing clinical practices, which are primarily based on measurements under “static” conditions. Thus, there is an emerging need for platforms that allow direct visualization and mapping of cytokine production, diffusion, transportation for better understanding the highly heterogeneous functional diversity of polyfunctional CAR T-cells and immune cell communications. To address this need, the central objectives of this proposal are to develop novel integrated `nanoplamson ruler'-based nanosensing technology to resolve the temporal dynamics of cytokine secretion from individual CD19 CAR T-cells and the crosstalk with B-ALL cells and bone marrow immune suppressor cells. The success of this technology will allow, for the first time, the direct visualization of multiplex cytokine secretion from individual CAR T-cell in a high-sensitivity, multiplex, label-free, in situ and real-time traceable manner. The proposed platform would provide a detailed and time-dependent mechanisms of how CAR T-cell response to stimulation and evolve in a suppressive niche for preclinical screening of optimal, effective and safe CAR T-cell therapy.

NIH Research Projects · FY 2025 · 2020-06

Project Summary This application seeks renewal of funding for an R25 that is building nationwide investigator capacity in optimization of behavioral and biobehavioral interventions. Multicomponent behavioral and biobehavioral interventions play a central role across many areas of public health, including but not limited to substance abuse disorders, HIV, cancer, and diabetes. To date, intervention science has relied primarily on the classical treatment package approach, in which a set of intervention components is identified a priori, assembled into a treatment package, and then immediately evaluated in a randomized clinical trial. Sole reliance on this approach has prevented the field from addressing fundamental research questions—e.g., which components are effective?—that are critical for development of interventions with sustained high public health impact. Recently an innovative alternative has emerged called the multiphase optimization strategy (MOST). MOST, a broad methodological framework for the principled optimization of interventions, is used to arrive at an intervention that strategically balances effectiveness against affordability, scalability, and efficiency to ensure that the intervention is practical to implement and therefore has the potential for sustained high public health impact. Between 2016 and 2022, annual NIH funding awarded for projects involving intervention optimization grew more than 450%, from about $30 million to about $144 million. At this writing, NIH has funded at least 245 projects related to MOST. We believe this growth has been enabled in part by the training efforts of the previous R25. We prepared and placed 2 free comprehensive asynchronous introductory courses on the Coursera platform, accessed by over 700 learners to date, and developed a synchronous virtual training aimed at helping investigators gain the skill set they need to write successful funding applications and conduct high- quality intervention optimization research. By the end of the first R25’s funding we expect more than 150 investigators will have completed our synchronous virtual training. In response to growing demand, we propose to expand our training, outreach, and support efforts. Aim 1: We will continue to offer synchronous virtual trainings, and coordinate with compatible existing NIH-sponsored training endeavors. Aim 2: In addition to updating our existing offerings, we will offer exciting new courses on the Coursera platform. Aim 3: We will enable investigators working with MOST to share the very latest findings, best practices, on-the-ground experiences, and tips with each other by offering 12-16 informal webinars per year. Aim 4: We will provide ongoing consultation, mentorship, support, and outreach for investigators at all career levels. Impact: The proposed work will increase the number of investigators proficient in and funded for optimization of behavioral and biobehavioral interventions. These investigators will, in turn, produce interventions that are effective, readily implementable, and sustainable, i.e., have high public health impact, and thereby improve the nation’s health in substance abuse, HIV, cancer, diabetes, and countless other important areas.

NIH Research Projects · FY 2026 · 2020-05

Project Summary/Abstract Childhood apraxia of speech (CAS) is a complex motor speech disorder that significantly limits a child’s ability to communicate in daily activities, with difficulties often persisting into adolescence and adulthood. There is solid evidence that motor-based interventions, such as Dynamic Temporal and Tactile Cueing (DTTC), improve word production in children with CAS. Building on this strong foundation, the next critical step is to extend this work to support functional communication in connected speech, where children often continue to struggle. Our work in the previous funding cycle showed that young children with CAS achieved significant gains in word accuracy and exhibited reduced articulator movement variability, underscoring DTTC’s powerful impact on speech motor control. However, without a systematic bridge to connected speech, even robust word-level gains may fail to generalize to other speaking contexts. For the next cycle of research, we will conduct a Phase II Randomized Control Trial (RCT) to study the efficacy of DTTC-Connect, a novel adaptation of DTTC that includes phrase-level practice to refine connected speech and support communicative participation for children with CAS. The overall objectives of this application are to test the efficacy of DTTC-Connect and document changes in speech motor control at the connected speech level. The central hypothesis is that DTTC-Connect will lead to lasting improvements in phrase accuracy, speech intelligibility and speech motor control, ultimately enhancing a child’s communicative participation. Aim 1 will quantify the effects of DTTC-Connect on connected speech accuracy and examine whether previous DTTC modifies response to DTTC-Connect in a subset of children who participated in the prior DTTC RCT. Aim 2 builds on our established methodology of kinematic and acoustic analysis to study the effects of DTTC-Connect on speech motor variability, while also exploring whether previous DTTC modifies changes seen in the speech motor system following DTTC-Connect. Lastly, Aim 3 will evaluate the effect of DTTC-Connect on communicative participation in children with CAS. DTTC- Connect represents a critical next step in maximizing clinical impact and advancing our understanding of speech motor learning in CAS. Together, these aims will provide a rigorous test of whether motor-based treatment can drive lasting, functional improvements in connected speech for children with CAS. This project bridges the gap between speech production gains and meaningful communication improvements, advancing both science and clinical care for children with CAS.

NIH Research Projects · FY 2026 · 2020-05

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Circadian clocks drive a range of rhythmic cellular outputs to control 24hr rhythms in behavior and physiology. These outputs include rhythms in cell size and shape, but the mechanisms are not well-understood. Circadian pacemaker neurons, hepatocytes and intestinal epithelial cells all show 24hr rhythms in cell size and shape, while fibroblasts show circadian rhythms in actin dynamics during wound healing. We study the circadian control of cell size and shape using the Drosophila s-LNv circadian pacemaker neurons. The structure of neurons makes them excellent cells to visualize changes in shape. In addition, the relative simplicity of the Drosophila brain permits visualization and quantification of s-LNv projections, and Drosophila genetics allows precise spatial and temporal manipulation of s-LNv gene expression. We plan to study 3 areas that address key aspects of control of circadian changes in cell shape. Growth factors and other signals induce immediate early gene (IEG) transcription in most cells. Neuronal firing is typically the relevant signal for IEGs in neurons. In Area 1, we will study a transcriptional program activated by the opposite signal in s-LNvs: absence of neuronal activity. Neuronal inactivity activates transcription of toy, which encodes the fly Pax6 transcription factor. Toy then activates transcription of Pura, which encodes a GEF that increases Rho1 activity to retract s-LNv projections via the actin cytoskeleton. Mutations in Pura that prevent s-LNvs from changing shape cause long period behavioral rhythms, underlining the importance of rhythmic s-LNv shape changes for accurate 24hr rhythms. We will dissect the inactivity-regulated gene expression pathway, and test if it functions in different cells – including broadly across the brain during sleep. This gene regulatory network may also be activated when growth factors are removed from fibroblasts to ensure they do not progress through subsequent cell cycles. In Area 2, we focus on the function of Pura as a GEF. Pura activates Rho1, which in turn activates effectors including Rho Kinase. However, Pura itself is an in vitro target of Rho Kinase. Post-translational regulation of Pura could function either in a feedforward loop to lock Pura / Rho1 in an active state, or via negative feedback to limit the activity of Pura / Rho1 and ensure that s-LNv projections do not over-retract. We will test these ideas in vivo in s-LNvs. Our findings should have broad implications for control of Rho family GTPase activity, which is important in processes such as mitosis, cell movement and phagocytosis as well as cell morphology. Area 3 focuses on connections between cells: How do cells know which other cells to interact with? This question is of general importance, but is especially important in the nervous system. We will build on our novel connectomics assay to understand the cell adhesion molecule code that s-LNvs use to make and break connections with downstream cells as they change structure.

NIH Research Projects · FY 2026 · 2020-04

The ability of some organisms to regenerate damaged body parts long after embryogenesis is a fascinating and critically important property. The remarkable capacity of plants to regenerate from almost any organ arises from their ability to recreate its organ-specific growth centers, known as meristems. Plant regeneration provides a unique model to learn how cells communicate during regeneration because plant cells are non-motile and must rely on cell-cell signaling to enable complex tissues to reform. In addition, plant regeneration has practical implications for human nutrition, as modern biotechnological techniques to improve crops rely on tissue regeneration. This MIRA renewal explores mechanisms that make up the plant’s self-organizing system that forms new organs during regeneration. Building on the prior work generated by the MIRA award (R35GM136362), the project uses techniques that uncover the wide repertoire of mobile transcription factors, microRNAs, and peptides that mediate pattern formation during regeneration. The technique uses inducible constructs that block communication from specific cells and then reads out the effect of the communication block in single-cell RNA-seq profiles, called Block-Seq. This renewal also embarks on the functional analysis of mobile signals, using highly parallel pooled CRISPR/single cell RNA-seq screens in whole plants to identify mutations with cell non-autonomous effects. In a second project related to signals in plant regeneration, this MIRA renewal examines how bursts of glutathione in the nucleus instigate rapid cell divisions that characterize the early steps of regeneration. The project investigates whether fast divisions, which prior results showed abbreviate the G1 phase of the cell cycle, are necessary to allow differentiated plant cells to reprogram after injury. In addition, the MIRA will open a third project to develop a model for regeneration that is a close relative of the most important crop plants, such as corn, rice, and wheat. Such a model will greatly aid in transferring regenerative properties to crop plants to speed the development of new crops that increase U.S. food security and improve the nutritional value of our diets. Overall, the project asks basic questions that will inform the wider field of regeneration and addresses a practical problem in plant biotechnology that directly impacts human health and nutrition.

NIH Research Projects · FY 2026 · 2020-03

PROJECT SUMMARY Collisions between the DNA replication and transcription machineries – known as Transcription-replication conflicts (TRCs) appear to occur in all eukaryotes. Although these conflicts have been extensively investigated as a source of genome instability, we currently lack tools to determine how frequently these conflicts occur, where they are located in the genome, and whether the two complexes meet in a head-on or a co-oriented disposition. In addition to unwinding DNA to generate two single strands to serve as the template for replication, DNA helicases play diverse roles in DNA replication and repair. Mutations in genes encoding helicases are implicated in a number of human diseases including but not limited to cancer. A major limiting factor in our understanding of helicase function in cells is an inability to precisely determine the sites at which they are active. The proposed work encompasses two ongoing projects: Existing methods to map protein-DNA interactions do not distinguish sites at which a helicase is close to DNA from those at which it is actively unwinding. The first project takes advantage of a novel strategy to map helicase activity on genomic DNA via fusion to a single-strand-specific cytidine deaminase. Using this strategy, we will define the targets of Pif1-family helicases at high resolution, investigate the degree to which partially redundant helicases can compensate for one another, and determine which sites each helicase targets during each phase of the cell cycle. This work will also establish pipelines that will allow future investigation of other helicases and single-stranded DNA binding proteins in unicellular and multicellular eukaryotes. The second project will advantage of an optimized split-enzyme proximity biotinylation system to map genomic loci at which replication and transcription complexes physically interact. Among other experiments, we will test a long-standing model wherein replication forks can bypass transcribing RNA polymerases without evicting them, allowing for the resumption of transcription without loss of the nascent RNA transcript. The core DNA replication and transcription machineries, as well as most helicases, are highly conserved throughout eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of fundamental biological processes. Therefore, the results of this work will provide molecular insights into genome instability in humans; these results will be directly applicable to our understanding of the etiology and progression of cancer, as well as diseases associated with impaired helicase function.

NIH Research Projects · FY 2024 · 2019-09

Oral cancer patients suffer severe chronic and mechanically-induced pain. Opioids are initially effective, but dose escalation is required and side effects reduce quality of life. The long-term goal is to improve management of oral cancer and oral cancer pain. Oral cancer pain is initiated and maintained in the cancer microenvironment. Some overexpressed cancer genes, oncogenes, can function in an autocrine manner to promote cancer and in a paracrine manner as cancer pain mediators. The ensemble of altered genes/pathways in a cancer dictates response to treatment, which motivates the use of combinatorial therapies tailored to the individual (precision medicine) to both treat the cancer and pain. The overall objectives of this application are to determine (a) whether artemin (ARTN), a gene overexpressed in oral cancer is an oral cancer oncogene, (b) whether ARTN is an oral cancer pain mediator and (c) whether antagonizing ARTN stops oral cancer and alleviates oral cancer pain. The central hypothesis is that there are oral cancer oncogenes that promote cancer and induce oral cancer pain. The rationale for this project is that proalgesic oncogenes could be targeted to treat cancer and pain. The central hypothesis will be tested by pursuing three specific aims: (1) Determine if ARTN is a proalgesic oncogene in human cancer; (2) Determine whether ARTN is an oncogene and a nociceptive mediator; and (3) Determine the potential to stop oral cancer and alleviate oral cancer pain by antagonizing proalgesic oncogenes. In the first aim, expression of ARTN will be assessed by immunohistochemistry in archival specimens from patients who completed the UCSF Oral Cancer Pain Questionnaire (UCSFOCPQ) to determine if expression is correlated with pain. The second aim will evaluate the function of ARTN as an oncogene by manipulating expression in cultured cells in vitro and in human xenograft mouse models. Whether ARTN is a pain mediator will be assessed by measuring nociception induced by manipulating expression of ARTN in animal models in the absence of cancer growth. For the third aim, the potential of antagonizing ARTN to stop cancer and cancer pain will be evaluated by anti-ARTN treatment of mouse xenograft and carcinogenesis models. The proposed research is innovative in the applicants' opinion, because it uses information gained from genomic analysis of oral cancers to identify putative oral cancer proalgesic oncogenes. The research is significant because it is expected to lay the foundation for future clinical trials assessing the utility of targeting ARTN for cancer treatment and attenuation of cancer pain. The work will motivate identification of additional proalgesic oncogenes to improve precision cancer pain management.

NIH Research Projects · FY 2025 · 2019-08

Project Summary/Abstract Our laboratory is primarily focused on elucidating the molecular mechanisms integral to transmembrane receptor function and modulation of their signaling output. We employ cryo-electron microscopy (cryo-EM) in conjunction with advanced classification methodologies such as manifold embedding, modeling, and molecular dynamics to investigate how ligand binding influences conformational equilibria and signaling bias. Our research encompasses various systems, enabling us to examine the impact of different ligand types on ion channel gating and G protein-coupled receptor (GPCR) activation. Our longstanding research interest lies in receptors vital to heart and skeletal muscle function. We are investigating the mechanisms and modulation of ryanodine receptors (RyR) and beta-adrenergic receptors, which play a crucial role in cardiac and skeletal muscle functionality. Our objective is to elucidate how small molecules, protein regulators, and ions shape the conformational energy landscape of these receptors. This knowledge is critical for understanding calcium signaling regulation and for designing innovative therapeutics to address cardiac and muscle disorders. Another research interest is exploring lipid-triggered gating of mechanosensitive channels from the MscS family, a model system for membrane tension-sensing. This research can provide valuable insights into the fundamental principles governing ion channel gating and the role of lipid-protein interactions in this process. Our research approach combines cryo-EM, advanced image classification techniques, residue network analysis, and molecular dynamics simulations to mechanistically delineate allosteric pathways. We subsequently validate proposed models using biophysical techniques such as single-channel measurements, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and mutagenesis. Our long-term goal is to significantly enhance our molecular understanding of receptor activation and allosteric modulation. Advancement in this field could potentially pave the way for the design of small molecule allosteric modulators with precise control over their effects on their targets, thus aiding in the development of drugs with minimized side effects. 1

NIH Research Projects · FY 2026 · 2019-08

Research summary Regulation of transcription is essential for cell function. In eukaryotes, diverse mechanisms control transcription through chromatin structure and organization of DNA within the nucleus. Histone modifications modulate the activity of gene regulatory elements and regulate binding of proteins that form transcriptionally active and inactive compartments. Structural maintenance of chromosomes (SMC) complexes bind and translocate on DNA, forming loops that bring distant sites into contact. As the molecular activities of individual chromatin modifiers and SMC complexes are being studied, it is important to determine how they integrate with each other to control transcription during development and differentiation. Our research program addresses the functional interactions between SMC complexes, chromatin, and transcription around three topics. The first focuses on condensin, a eukaryotic SMC complex that has been challenging to study in vivo because it is essential for cell division and binds chromosomes transiently during mitosis. In the nematode Caenorhabditis elegans, a hermaphrodite-specific condensin functions throughout the cell cycle to repress X chromosome transcription for dosage compensation. Condensin DC also controls histone modifications, providing an excellent system to study integration of SMC activity and chromatin modifiers for transcription regulation. The second topic focuses on the interaction between condensin and cohesin, another SMC that regulates the organization of eukaryotic genomes during interphase. As cells progress from interphase to mitosis, cohesin binding and activity decreases while condensin’s increase and transcription is silenced. This transition is key to regulation of cell-type specific transcriptional programs during differentiation. By addressing how condensin DC interacts with cohesin on the X chromosomes, we believe our work can provide insights into the interphase to mitotic transition of the eukaryotic genomes. The third topic is regulation of RNA Polymerase III, which transcribes a diverse array of small functional RNAs, including tRNAs. RNA Pol III regulation is relatively understudied and is important for protein translation in health and disease. Due to unique chromosomal distribution of tRNA genes, our work in C. elegans dosage compensation puts us in an ideal place to study the mechanisms that regulate RNA Pol III transcription. In the next five years our research will provide significant insights into how cohesin, condensin and chromatin work together to regulate transcription during development and differentiation. Our program is also evolving to address environmental gene regulation and RNA Pol III transcription – new and important areas of study to understand basic mechanisms of genome organization and function throughout the life of an organism.

NIH Research Projects · FY 2025 · 2019-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The widespread adoption of genome-scale methods in biomedical research has resulted in biology now being a big data discipline that requires training in modern machine learning and artificial intelligence (AI). Formal instruction in both biology and AI is critical for PhD trainees pursuing careers in a broad range of academic and non-academic fields. The mission of the Quantitative Biological Systems Training (QBIST) program is to empower biomedical researchers to choose confidently amongst a range of careers through active training and experiential learning in robust and transferable quantitative and leadership skills. The second award period of the QBIST program will focus on 3 primary objectives. Objective 1: Consolidate training opportunities to develop and apply advanced computational and data science skills to complex biomedical research questions. We will modify the QBIST curriculum to respond to the evolving demands of quantitative training to ensure training in cutting edge methods. Objective 2: Enhance the value of mechanisms for exploring biomedical career paths outside the traditional academic trajectory. QBIST trainees will continue to explore post-graduate career options through participation in an internship program with non-academic organizations. In addition, we will provide opportunities to participate in established entrepreneurial and multi-dimensional team-building programs at NYU to expand training options.  We will also increase the value of these experiences by providing opportunities for self-reflection. Objective 3. Provide trainees with opportunities to develop leadership skills through mentoring experience. To enable trainees to gain critical leadership and mentoring skills trainees will assume mentorship roles in the NYU Biology Summer Undergraduate Research Program. Importantly, we will modify the existing QBIST curriculum so as to not increase trainee workload to ensure we meet our overall objective of decreasing time to graduation while maintaining a high PhD graduation rate. The second award period of the QBIST program will include 17 faculty members at all career stages with a demonstrated track record of mentoring and commitment to the goals of the QBIST program. To accommodate the expanded aims of the QBIST program we will select 4 students per year who will be appointed for two years during the second and third years of their PhD training. Students will be selected by the QBIST program executive committee on the basis of a written application detailing training goals and long-term career objectives that demonstrate alignment with the QBIST program. To assess the continued effectiveness of the QBIST program, we will use annual surveys completed each year by all trainees and quantify trainee research and career outcomes. All data and analyses will be made publicly available on the QBIST web portal. Achieving the QBIST program objectives will serve to enhance the entire NYU Department of Biology PhD program and provide insights and resources that will inform PhD training in biomedical institutions throughout the USA.

NIH Research Projects · FY 2025 · 2019-04

Project Summary Late-stage modification of pharmaceutically relevant small molecules and biomolecules, including alcohols, peptides, and carbohydrates, is crucial in drug discovery and chemical biology research. However, achieving selectivity and specificity among the diverse functional groups present within such molecules is a primary challenge in late-stage functionalization. This proposal aims to address this challenge by leveraging the non- polar nature of radical intermediates, which exhibit orthogonal reactivity compared to the prevalent polar functional groups found in biomolecules. Specifically, a phosphoramidite reagent will selectively functionalize hydroxyl groups by enabling deoxygenative radical formation and incorporate it into various transformations, including the Giese reaction, cross-coupling, and trifluoromethylation reactions. The mild conditions and moderate electrophilicity of the phosphoramidite will offer compatibility with biomolecules rich in various reactive groups. Mechanistic studies of radical capture by nickel intermediates will inform catalyst design for stereoconvergent deoxygenative coupling of alcohols. Furthermore, understanding this fundamental step will provide insight for the design of a broader range of cross-coupling reactions involving radical intermediates generated from various means, including chemical, photoredox, and electrocatalytic activation. Finally, the proposed research aims to develop novel photoredox electron donor-acceptor coupling reactions that facilitate the synthesis of cyclic peptides, a promising strategy for drug development. These reactions will expand the chemical space accessible to drug discovery and facilitate the development of new therapeutic agents.

NIH Research Projects · FY 2026 · 2019-02

Abstract Interactions of proteins with other biomolecules regulate fundamental cellular events and misregulation of these interactions leads to disease states. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these folded domains we can specifically inhibit chosen protein complex formation with rationally designed synthetic molecules. Based on this hypothesis, we have developed a suite of Protein Domain Mimics (PDMs) that faithfully reproduce binding epitopes on protein surfaces. This work has created a foundation for the development of a new class of structure–based therapeutics. Equipped with our platform of PDMs, we will focus on a currently intractable class of targets in IDPs or Intrinsically Disordered Proteins. Therapeutic targeting of intrinsically disordered proteins is attractive because they interact with a multitude of partners and influence numerous signaling pathways. However, targeting of IDPs remains underexplored. We hypothesize that we can engage cellular IDPs with biomolecular receptors and scavenge them away from their natural binding partners. This strategy would constitute a distinct mechanism for targeting of IDP-mediated protein-protein interactions. In a new direction for the group, we will develop encodable ligands to sequence-specifically target double-stranded RNA, the most abundant class of cellular RNA. dsRNA has proven to be recalcitrant to therapeutic intervention; although, it is central to many biological events. In preliminary results, we have discovered a new class of molecular scaffold that can be engineered to provide sequence-specific recognition of dsRNA. Studies in each Aim will advance general approaches to inhibit protein-protein and protein-RNA complexes, and establish PDMs as distinct constructs spanning the molecular size space between small molecules and proteins.

NIH Research Projects · FY 2026 · 2019-01

Project Summary/Abstract Children with speech sound disorder show diminished intelligibility in spoken communication, with negative con- sequences for both social-emotional and academic-occupational outcomes [1–4]. While most speech deviations resolve by the late school-age years, between 2-5% of speakers exhibit residual speech sound disorder (RSSD) that persists through adolescence or even adulthood [5–7]. Both affected children/families and speech-language pathologists (SLPs) have highlighted the critical need for research to identify more effective forms of treatment for children with RSSD. Our work in the previous funding cycle showed that individuals with RSSD benefit from treat- ment incorporating technologically enhanced sensory feedback (visual-acoustic biofeedback, ultrasound biofeed- back). However, real-world adoption of biofeedback treatment remains limited by equipment costs and lack of access to providers with specialized training. For our next phase of research, we focus on developing methods to support wider implementation of technology-enhanced treatment for RSSD. Specifically, we investigate the possibility that access to biofeedback can be expanded through telepractice service delivery and the use of Artificial Intelligence (AI)-powered technol- ogy to extend the services provided by SLPs. At the same time, we evaluate whether the efficacy of biofeedback can be further enhanced by adopting a precision medicine approach in which a treatment method is selected based on a learner’s individual profile of sensory strengths and weaknesses. In this proposal, we will conduct the first controlled comparison of biofeedback treatment delivered in person versus via telepractice (Aim 1), testing our hypothesis that biofeedback intervention can be delivered remotely without any unacceptable loss of efficacy. If successful, this study will greatly expand the reach of biofeedback by making it easier for children with RSSD to connect with trained clinicians. In Aim 2, we will utilize technology de- veloped in the previous funding cycle to assess whether the maintenance of gains achieved through biofeedback treatment can be enhanced through AI-mediated home practice. Finally, Aim 3 will lay groundwork for a precision medicine approach by testing whether relative response to ultrasound and visual-acoustic biofeedback can be predicted from a learner’s profile of sensory response across auditory and somatosensory domains. While new technologies have the potential to revolutionize the development and delivery of interventions for speech disorders, there is an ongoing need for well-designed research studies to bring these advances into evidence-based clinical practice. This research will address the needs of children with RSSD and the clinicians who treat them by providing a robust evidence base to guide clinical decision-making and user-friendly tools that support implementation.

NIH Research Projects · FY 2026 · 2018-09

Project summary: Ants are social insects that live in colonies of morphologically and physiologically different individuals that are essentially identical genetically, making ants an attractive system to study epigenetic phenomena. Ant colonies contain many workers that perform most tasks but do not lay eggs, while queens are solely responsible for reproduction. Remarkably, queens live up to 10X longer than workers, in sharp contrast with most animals in which high reproduction leads to shortened lifespan. The jumping ant Harpegnathos saltator exhibits a high degree of aging plasticity: In the absence of the queen, some workers can become pseudo-queens called gamergates. Gamergates dramatically change their behavior, produce eggs, reconfigure their brain and most dramatically, have a 5X lifespan extension. Remarkably, when placed in the presence of a genuine queen, gamergates transition back into workers with an accompanying shortened lifespan. We established Harpegnathos as a model system that can be manipulated with CRISPR/Cas9, providing a unique opportunity to study the molecular mechanisms that control aging, as well as the crosstalk between aging and reproduction. Using a combination of transcriptomics as well as both ex vivo and in vivo pharmacological manipulations, we discovered that gamergates have an elevated production of Insulin accompanied by differential regulation of the two branches of the Insulin signaling pathway (IIS) in target tissues. The MAPK branch of IIS is activated in the gamergate fat body and ovary, while the AKT branch is repressed by extracellular “anti-Insulin” proteins, ImpL2. As MAPK activity is required for egg-laying, we hypothesize that repression of the AKT branch contributes to the dramatically extended longevity in gamergates. We now propose to investigate the molecular mechanism of ImpL2 function and test its role in aging. First, we will identify the source and organismal distribution of ImpL2, and then experimentally modulate its levels and mutate ImpL2 to examine the effect(s) on IIS and increased metabolism for egg formation. Furthermore, we will explore the molecular interactions of ImpL2 and the mechanisms that lead to the specific inhibition of the AKT (but not MAPK) IIS pathway. Next, we will test the effect of ImpL2 on aging in manipulated animals, assessing a panel of aging biomarkers and extend our investigations to another anti-Insulin protein, ALS. Moreover, tissue- specific manipulation of ImpL2 expression in Drosophila will address its potentially conserved effect on reproduction and lifespan in a powerful model system. Finally, we will extend our study to the brain remodeling events that accompany and orchestrate the social transition. We will perform single-cell mRNA sequencing of the different social groups/ages to survey age-associated changes in the Harpegnathos brain and identify candidate regulators responsible for delayed aging in gamergates. We will exploit the transcriptomic data to test how specific genetic functions modulate brain circuits and aging.

NIH Research Projects · FY 2026 · 2018-05

Abstract The overall goal of my research program is to develop and apply state-of-the-art machine learning and molecular modeling tools to facilitate the rational design of modulators of important cellular pathways for therapeutic use. Protein-protein interactions (PPIs) are central factors in cellular signaling and biological networks, and their mis-regulations lead to diseases states. Thus PPIs are biologically compelling targets for drug discovery. Despite a few notable successes, most PPIs have not been successfully targeted and remain challenging for therapeutic intervention. The fundamental challenge derives from their intrinsic structural features: the binding surfaces of many PPIs are generally large in area, flat, and dynamic. PPIs are often transient and involve multivalent contacts. One of the most promising PPI inhibitor discovery strategies is to use miniature protein domain mimetics (PDMs) to reproduce the key interface contacts utilized by nature. PDMs are advantageous as medium-sized molecules with high surface complementarity and a broader set of contact points than typical small molecules, but are still limited because—by definition—only a portion of the total PPI binding energy is captured in the interaction. The binding affinity of the synthetic domains is often lower than the cognate full-length proteins. In last five years, we have significantly advanced a pocket-guided rational design approach based on AlphaSpace to tackle this challenge. We have successfully optimized a PDM to target the KIX domain of the p300/CBP coactivator by introducing non-natural amino acids to improve pocket-fragment binding; rationally designed a novel NEMO coiled coil mimic that disrupts virus-induced NF-κB signaling and induces cell death; and successfully targeted a new binding pocket on MDM2 and MDMX with a potent dual inhibitor by elaborating hydrogen-bond stabilized alpha-helix mimetics. Meanwhile, we have developed state-of-the-art scoring functions for protein-ligand docking as well as virtual screening, advanced deep learning models to predict molecular properties and chemical reactions, and established strong and fruitful collaborations with several outstanding experimental labs in chemical biology and biophysics to discover new modulators of biomolecular interactions. These significant advances set the stage for us to further push the frontier of integrating machine learning and molecular modeling for rational drug design. Our focus in the next few years will be to establish a robust pocket-guided design platform based on AlphaSpace and machine learning for PPI orthosteric inhibitor optimization, provide physical/chemical insights and develop novel computational strategies for allosteric modulator discovery, and explore chemical space with deep sequence/graph/geometric representation learning for multi-objective molecular design. Our modulator design efforts in close collaborations with our experimental colleagues will not only rigorously test predictive power of our developed methods in real life applications, but also result in highly specific and potent modulators towards several important but challenging therapeutic targets, providing new leads for drug development.

NIH Research Projects · FY 2025 · 2018-05

Abstract Accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR) which aims at restoring a healthy cellular proteome. Dysregulation of the UPR is key to diseases, e.g. neurodegeneration. For this central role, the UPR forms a network of processes, involving complex transcription, translation, and RNA and protein degradation changes. For example, while the UPR shuts down global translation, it activates specific response genes, such as the transcription factor ATF4. Our goal is to investigate this response network. We have investigated this network from several angles: profiling the dynamics of the mammalian UPR, we identified regulatory signatures for hundreds of genes. We discovered a translation regulatory element in ATF4 whose role in translation induction of the gene under stress had been overlooked. The element consists of a start and stop codon and stalls ribosomes. We discovered start-stops in hundreds of genes enriched for signaling molecules. In addition, we profiled protein modifications, e.g. ubiquitination, in response to stress. Further, we began to compare the UPR in two closely related motor neurons with differential stress-sensitivity that is consistent with their role in Amyotrophic Lateral Sclerosis (ALS): stress-sensitive spinal motor neurons die early during ALS, while more stress-resistant cranial motor neurons survive until late stages of the disease. We identified molecular signatures, e.g. in the proteasome, that can explain the motor neurons’ differential stress sensitivity. Doing so, we created tools and resources for integrative analysis. In the next five years, we will address three major questions that arise from these findings: i) How does the cell, in general, induce stress response genes while general translation is halted? ii) What are the roles of protein modifications in the UPR? and iii) How do translation, protein modifications, and other pathways form an efficient and robust response network that restores proteome health upon stress? Specifically, we will investigate the role of start-stops and other elements in translation regulation of DROSHA and RAD23B, which function in the miRNA pathway and DNA damage repair, respectively, but also link to the ER stress response. Using a gain-of-function construct, we will deconvolute the mechanism of start-stop function and identify regulators of ribosome stalling that affect transcript localization, stability, and downstream re-initiation. We will complement these analyses with large-scale assessment of ribosome scanning and initiation, changes in transcript stability and in proteins bound to mRNAs in response to stress. A second research avenue will investigate protein ufmylation, a ubiquitin-like protein modification linked to the UPR and ER maintenance, but also to translation and the DNA damage response. We previously found that Ufl1, a key ufmylation gene, expresses different isoforms in cranial and spinal motor neurons, and our proposed work will investigate the impact of differential Ufl1 expression on the UPR in the two motor neurons. Complementing these analyses, we will attempt to identify novel and neuron type specific ufmylation targets and investigate their links to translation regulation, ribosome quality control, and DNA damage repair. The work will exploit our expertise in systems-scale and targeted analysis to understand new properties of the proteostasis network.

NIH Research Projects · FY 2025 · 2017-09

Project Summary Vision at the center of gaze (fovea) has high sensitivity and resolution, facilitating good performance in many tasks. But performance worsens with increasing distance from fovea–eccentricity. At any given eccentricity stimuli can fall anywhere along the circle –polar angle. Both eccentricity and polar angle have pronounced effects on perception in human adults. These factors present an ideal opportunity for establishing tight quantitative links between behavior and neural representations of visual information. Our long-term goal is to understand how visual performance varies across the visual field, to develop a theory of spatial vision that includes the neural and computational mechanisms underlying performance variation with eccentricity, polar angle and individuals. Such a theory will be applicable to basic and translational research in perceptual and cognitive neuroscience. We propose to investigate whether and how neural and computational factors distinctly limit discriminability across observers, eccentricity and polar angle. Our overall hypothesis is that variability in cortical magnification limits discriminability as a function of eccentricity, polar angle and observer, and that these effects are mediated by different combinations of noise, efficiency and sensory tuning. The proposed psychophysical [Aim 1] and neuroimaging [Aim 2] measures will characterize internal/neural noise and sensory/neural tuning across eccentricity and around polar angle, and will constrain a computational observer model of contrast sensitivity and acuity tasks [Aim 3]. In addition to advancing our knowledge of visual perception and cognitive neuroscience, the proposed research will enable us to make predictions about human performance. The characterization of eccentricity and polar angle has significant implications for ergonomic and human factors applications as well as for public health. For example, it is of critical importance for user-interfaces that present information at different locations of the visual field. We can extend our knowledge to real-world displays, such as navigation and cockpit alerting systems, control panel layouts in cars, computer-aided detection systems, and software for presenting radiological images. Furthermore, the gained knowledge can aid the design of artificial image recognition systems. In addition, understanding the underlying neural and computational mechanisms of performance differences across the visual field will improve our models of visual dysfunction (e.g., macular degeneration, retinitis pigmentosa), as well as the diagnosis of these disorders.

NIH Research Projects · FY 2026 · 2017-09

Abstract As advances in both experimental and computational biology lead to exciting discoveries in many fields of biology and biomedicine today, new avenues to diagnose and treat human disease are becoming a reality. Molecular dynamics and other simulation approaches play a key role in these connections by helping define the underlying biophysical mechanisms, at unprecedented resolution. The PI's computational biophysics lab focuses on solving fundamental structural and functional challenges concerning nucleic acids and their complexes (notably chromatin and RNA), in collaboration with experimentalists, by innovative molecular models and simulation methods using ideas from mathematics, computer science, engineering, biology, and chemistry. This MIRA project would continue to advance our fundamental understanding of genome organization and RNA motifs using multiscale models that bridge disparate spatial and temporal scales to generate biophysical insights into genome folding and cancer genomics, and RNA conformational landscapes/ viral mechanisms. For chromatin and chromosomes, modeled nucleosomes and their protein complexes at atomic resolution, and chromatin fibers at the mesoscale, will be linked to data from genome-wide and cancer epigenomics studies to determine the modulation of chromatin higher-order structure in processes of aberrant gene expression. Specifically, we will focus on the structural role and mechanisms of proteins (like CTCF and H1) in gene looping and formation of topologically associated domains (TADs) and nuclear compartments in gene activation or repression in cancer cells. For RNA, our topological approach to modeling RNA secondary structure by coarse-grained graphs (RAG: RNA-As-Graphs), combined with atomic biophysical modeling, will delineate programmed ribosomal frameshifting mechanisms in RNA coronaviruses and other viruses, and advance the RNA motif atlas. Specifically, conformational dynamics associated with the RNA frameshifting element of SARS-CoV-2, along with evolutionary and biophysical analysis and chemical reactivity experiments, will describe frameshifting transitions and identify/test experimentally structure-altering mutations that may hamper frameshifting. RAG will also be applied to define a virus topology atlas and explore virus motifs from an RNA repertoire point of view, to help understand the RNA motif universe and advance novel RNA design. The unraveled biophysical mechanisms in genome organization and RNA frameshifting/conformational repertoire have translational ramifications for human cancers and viral infections by coronaviruses or HIV. For cancer therapy, a targeted re-expression of silenced genes may be possible by chromatin topological changes (e.g., loop dissolution) and RNA editing. For viral RNA infections, new strategies for gene and anti-viral therapy emerge from this research, feasible by modern RNA editing technologies. The resulting multidisciplinary computational paradigms are widely applicable to other biomolecular processes and will be shared with the community at large.

NIH Research Projects · FY 2026 · 2017-08

PROJECT SUMMARY/ABSTRACT G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are mediators of and therapeutic targets for diverse disorders, including chronic pain. This proposal challenges the dogmas that these receptors signal primarily from the plasma membrane and that cell surface receptors are the optimal therapeutic targets. Completed studies revealed that GPCR endosomal signaling controls pain and that endosomally-targeted antagonists provide more effective analgesia than conventional drugs. The renewal application extends these concepts to tropomyosin receptor kinase A (TrkA), a RTK for nerve growth factor (NGF). Although successful for the treatment of pain, NGF monoclonal antibodies have not been approved due to adverse outcomes of systemic antagonism. A deeper understanding of how NGF and TrkA signal pain is required. Anatomical and electrophysiological studies with mouse and human nociceptors, behavioral studies in mice, biophysical assays using recombinant proteins, model cells and nociceptors, and nanoparticle-encapsulated antagonists will be used to study NGF pain. Aim 1 hypothesizes that neuropilin 1 (NRP1) is required for NGF-induced sensitization of nociceptors and NGF-evoked nociception. Preliminary studies suggest that NRP1 and the scaffolding protein GAIP/RGS19-interacting protein (GIPC1) are necessary for NGF-induced nociception. Co- expression of TrkA, NRP1 and GIPC1 protein and mRNA in mouse and human nociceptors will be studied. Inhibitors of NRP1 and GIPC1 will be used to ascertain their contributions to NGF- induced sensitization of nociceptors and nociception. Aim 2 hypothesizes that NRP1 acts as an NGF coreceptor and TrkA chaperone to enhance NGF signaling of pain in nociceptors. Biophysical approaches will be used to study NGF association with NRP1, assembly of TrkA/NRP1 heteromers, TrkA surface expression, and NGF signaling in subcellular compartments of nociceptors. The role of GIPC1 as a scaffold for TrkA and NRP1 association will be studied. Aim 3 hypothesizes that endocytosis and endosomal NGF/TrkA/NRP1 signaling in nociceptors mediates sustained sensitization and nociception. The contribution of endocytosis to NGF-induced nociception will be studied using inhibitors of endocytosis and NGF/TrkA/NRP1 antagonists encapsulated into nanoparticles designed to deliver cargo to endosomes of nociceptors. NGF-induced endocytosis of TrkA/NRP1, compartmentalized signaling, nociceptor sensitization and nociception will be studied. A deep understanding of TrkA trafficking-dependent signaling will provide insights into the mechanisms and treatment of chronic pain, with implications for other RTK-mediated pathologies (e.g., cancer).

NIH Research Projects · FY 2024 · 2017-04

ABSTRACT The calvaria (upper part of the skull) comprises plates of bone and fibrous joints (sutures and fontanels). While the bone protects the brain, the sutures contain stem cells for osteoblasts, and thus allow the skull to grow coordinately with the expanding brain of a child. Craniosynostosis (a premature loss of the suture(s)) is a major class of human birth defects. It can lead to dysmorphic skull, and further affect brain and orofacial development. Current treatment for craniosynostosis often involves invasive and repetitive surgeries at young ages with relatively high rates of complications. Therefore, improving the methods of intervention for this defect is of great importance to public health. The calvaria is made of cells from the neural crest and the mesoderm in embryos. In mice, these cells form a mesenchyme layer that completely encases the brain soon after mid-gestation (cranial mesenchyme). The cranial mesenchyme on the apical side of the head (a.k.a. early migrating mesenchyme, EMM) gives rise to soft tissues such as the sutures, the dermis/hypodermis of the scalp, and the meninges. Our previous study has shown that LMX1B (LIM homeobox transcription factor 1b) plays a key role in suppressing osteogenesis in EMM to allow suture formation. Recently, single cell sequencing and in silico analyses from our group and others have suggested that EMM contains two populations of intermediate progenitors: suturodermal progenitors (SDP) with a dual potential for the suture mesenchyme and the dermis/hypodermis, and common meningeal progenitors (CMP), which contribute to the dura and the arachnoid layers of the meninges. We found that both SDP and CMP populations were severely decreased in Lmx1b mutants, whereas cells with a restricted fate were increased and appeared precociously. The goal of this project is to identify factors underpinning cell fate specification and differentiation in the cranial mesenchyme, and to utilize this information to generate SDP in vitro from human embryonic stem cells (hESC). In Aim1. we will determine transcription factors and signaling pathways that regulate cell fate specification and differentiation in the cranial mesenchyme in vivo. In Aim2, we will determine genetic regulators that control SDP specification and differentiation in vitro from hESC. The completion of this project will fill the critical gap in the current knowledge of suturogenesis. Furthermore, our results can facilitate efforts to use suture stem cells for craniosynostosis treatment, a promising new direction of research in the field.