Grants

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The mammalian heart undergoes profound transcriptional and phenotypical remodeling during postnatal development, a process known as cardiac maturation. However, the molecular mechanisms driving this transition is not fully understood, posing a major challenge in cardiac regenerative medicine, where induced cardiomyocytes from pluripotent stem cell differentiation or non-myocyte reprogramming exhibit an overall immature phenotype that severely limits their application in cell therapy and in vitro disease modeling. In this K99/R00 application, I propose to integrate cutting-edge single cell multiomics with state-of-the-art computational methods to unravel the cell-type-specific gene regulatory networks governing cardiac maturation, and develop a novel dual-reporter system to model and enhance cardiac maturation in vitro and in vivo. During the K99 phase, I will characterize the epigenomic changes of the mouse heart during postnatal development at a single cell resolution using various single cell multiomic technologies (Aim 1), and construct cell-type-resolved gene regulatory networks underlying cardiac maturation using bioinformatic approaches coupled with deep learning (Aim 2). I will also establish cell culture and mouse models with CRISPR-mediated knock-in of dual-fluorescent reporters to track and assess cardiomyocyte maturation (Aim 3a). During the R00 phase, I will experimentally characterize key regulatory elements and novel transcriptional regulators using functional genomic approaches (Aim 3b). I will also leverage these findings to enhance the maturation of in vitro-derived cardiomyocytes for improved therapeutic potential (Aim 3c). The expected outcomes of my proposed research will deepen our understanding of postnatal cardiac development and uncover new therapeutic strategies to improve cardiac function after injury. My career goal is to lead an independent research group that develops and employs innovative technologies to study the regulatory mechanisms underlying cardiac development, regeneration, and disease. In my K99 phase, I will acquire crucial knowledge and skills in advanced single cell genomics and computational biology to complement my previous expertise in developmental biology and cardiac research. My career development will be supported by an exceptional mentoring and advisory committee from UCSD/Salk/HHMI, along with world-class resources, training opportunities, and institutional support at UC San Diego. These elements will provide a strong foundation for my successful transition to an independent tenure- track faculty position.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary In mammalian cells, RNAs are predominantly located within cytoplasm and nucleus. A recent study, however, has reported that RNAs can be glycosylated, and such glycosylated RNAs (called glycoRNAs) are localized on out cell surface. The glycoRNAs are glycan-modified small non-coding RNAs. These RNAs are transcribed and maturated in nucleus and transported to cytoplasm for processing and then conjugated with glycan chains in endoplasmic reticulum. The conjugated molecules are then transported to cell membrane and anchored on cell membrane by binding to transmembrane RNA binding proteins. The cell surface glycoRNAs can serve as ligands or receptors to mediate cell-cell interaction, communication and infiltration and may also trigger cell signal transductions. Our recent works suggest that there were two forms of glycoRNAs, named glycoRNA-L and glycoRNA-S, robustly expressed in leukocytes, but not non-immune cells. GlycoRNA-L was migrated with ~11 kb RNA, whereas glycoRNA-S was migrated with ~0.6 kb RNAs. Using RNase removed the RNA frag- ments, the migration of both glycoRNA-L and glycoRNA-S was slightly faster, suggesting that the migration of glycoRNA-L and glycoRNA-S was determined by the glycan chains, but not RNA fragments. Further studies suggest that the glycan structures and functions of glycoRNA-L and glycoRNA-S may be distinguished. How- ever, the compositions and structures of glycan forms of glycoRNA-L and glycoRNA-S remain unknown. We hypothesize that the glycoforms of glycoRNA-L and glycoRNA-S from leukocytes are determinants of their functional significance. The goal of this proposal is to decode the compositions and structures of glycan forms of glycoRNA-L and glycoRNA-S from leukocytes. We will reach this goal by two approaches: 1) metabolic labeling combined with enzymatic digestion; 2) glycoRNA-L and glycoRNA-S purification and LC/MS analysis. Completion of the project will significantly advance our understanding of the role and mechanisms of glycoRNA- L and glycoRNA-S in the regulation of immune response and immune-related human diseases. The study will also provide a method for mapping the glycan structures of glycoRNAs from other cells such as cancer cells, which may provide insights for generating innovative diagnostic and therapeutic strategies.

NIA - National Institute on Aging

ABSTRACT Age-related cognitive decline and Alzheimer's disease are associated with hippocampal interneuron loss and hyperexcitability, which reflects the selective vulnerability of inhibitory neurons. Our preliminary data shows a ubiquitous reduction of inhibitory synapses onto hippocampal CA1 stratum radiatum dendrites in aged rats compared to young adults, regardless of their cognitive status. Aged rats that showed spatial learning deficits had enlarged excitatory synapses on these same dendrites, disrupting local E/I balance, which is important for learning and memory. In aged animals that maintained learning capacity, the remaining inhibitory synapses were larger, thus preserving local dendritic E/I balance. These data suggest that while some interneurons are vulnerable in the aging process, others are resilient and provide a potential compensatory role for maintaining local excitation and inhibition. The broad heterogeneity of interneurons reflects subtypes with various molecular profiles, firing rates, and differential targeting to specific CA1 dendrites. We hypothesize that specific interneuron subtypes are more susceptible to age-related changes, while others are resilient and contribute to compensatory learning mechanisms by maintaining local E/I balance. Studies that previously identified vulnerable interneuron subtypes in the aging brain typically used techniques to isolate one interneuron subtype at a time, limiting our understanding of how diverse interneuron populations interact and contribute to circuit function in the aging brain. Furthermore, the subcellular organelles and local resources crucial for ultrastructural plasticity, such as mitochondria, endosomes, smooth endoplasmic reticulum, and polyribosomes, remain largely uncharacterized in these interneuron subtypes during aging. Consequently, we lack a comprehensive understanding of how interneuron interactions and age-related alterations in their ultrastructural resources contribute to cognitive decline or resilience. This knowledge gap is exacerbated by limitations in traditional techniques for identifying interneuron subtypes that often compromise tissue quality, hindering ultrastructural analysis via serial section electron microscopy (3DEM). We propose using a novel ultraplex correlative light and electron microscopy (CLEM) method that allows for simultaneous identification of multiple interneuron subtypes via RNA and protein markers in tissue optimized for 3DEM. In Aim 1, we will use ultraplex CLEM to quantify interneuron subtypes and their subcellular structures in hippocampal tissue from behaviorally characterized aged rats, capturing any intrinsic ultrastructural changes that occur in these cell populations. In Aim 2, we will use our new machine learning tools to segment and reconstruct local interneuron circuits and synapses in distinct CA1 layers to identify alterations associated with age-related cognitive decline and resilience. This approach will provide valuable insights into the role of specific interneuron subtypes in maintaining E/I balance and cognitive function during aging, ultimately contributing to our understanding of selective neuronal vulnerability in Alzheimer's disease.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Post-transcriptional regulation is a critical mechanism controlling cellular differentiation and development, driven by coordinated interactions between RNA-binding proteins (RBPs) and RNA modifications. This proposal investigates the interplay between Quaking (QKI) isoforms and N7-methylguanosine (m7G) RNA modifications. QKI isoforms exhibit distinct subcellular localizations and functions, yet their roles in binding m7G-modified transcripts at 3′ untranslated regions (UTRs) during differentiation remain poorly understood. The central hypothesis is that QKI isoforms regulate differentiation by binding m7G-modified mRNAs at 3′ UTRs, with cytoplasmic QKI6 stabilizing transcripts essential for myeloid differentiation. Furthermore, m7G modifications may independently regulate gene expression in ways yet to be defined. Our preliminary evidence demonstrates that cytoplasmic QKI isoforms mediate myeloid differentiation, highlighting a critical gap in understanding m7G’s functional roles in steady-state cellular processes. Current methods for detecting m7G methylation at single- nucleotide resolution face technical limitations. This project addresses these challenges by employing direct RNA long-read sequencing and orthogonal methods to map m7G modifications with precision and identify QKI isoform-specific mRNA targets. Integrating these approaches will elucidate how QKI-m7G interactions influence myeloid differentiation, with broader implications for RNA modifications in stem cell biology, neurodevelopment, and cancer. Over the next five years, the laboratory’s mission is to (1) develop novel methods for base-resolution m7G detection, (2) define mRNA targets regulated by distinct QKI isoforms, and (3) determine the functional impact of QKI-m7G interactions on myeloid differentiation. These studies will advance understanding of RNA modification-driven gene regulation and may inform therapeutic strategies for diseases such as leukemia. By resolving the interplay between QKI and m7G at 3′UTRs, this work will reveal how their coordination regulates cellular functions across human cell types, directly addressing NIGMS’s mission to support foundational discovery science. The proposed research will provide mechanistic insights into RNA modifications dysregulated in cancer, potentially uncovering new targets for therapeutic intervention in malignancies and developmental disorders.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT The ubiquitin-proteasome system is a cornerstone of eukaryotic cellular regulation, orchestrating critical processes such as protein homeostasis, signal transduction, cell cycle progression, and stress responses. Central to this system are E3 ubiquitin ligases, which determine substrate specificity and mediate the transfer of ubiquitin from E2 enzymes to target proteins. This research focuses on unraveling the molecular mechanisms, structural dynamics, and regulatory networks of two highly conserved and essential classes of E3 ligases: Plant U-box (PUB) ligases and a specific family of RING ligases, with a particular emphasis on their roles under biotic stress conditions. U-box and RING ligases are conserved across eukaryotes, including humans, where their dysregulation is associated with various diseases. In plants and human U-box and specific RING ligases are integral to immunity, development, and stress adaptation, yet their precise biochemical mechanisms, including substrate recognition and regulation, remain poorly understood. Our preliminary studies have provided significant insights into these ligases. We determined the crystal structure of the U-box domain of a PUB E3 ligase in complex with E2 enzyme that represents a large and highly conserved class of E2s in eukaryotes, revealing a novel dimeric interface. We discovered a redox- sensitive disulfide bond switch in the E2 enzyme that dynamically modulates ubiquitin transfer and chain formation, both in vitro and particularly under biotic oxidative stress conditions. Using proximity labeling coupled with mass spectrometry, we performed distinct experiments including mapping the interactome of a PUB E3 ligase, and investigating the RING complex module under both normal and pathogen-exposed conditions. The experiment informed dynamic proteome changes in response to pathogen exposure, uncovering new relationships and previously unknown target substrates. Additionally, our CryoEM studies of the RING complex module provided critical insights into its dynamic substrate recognition mechanisms, proposing how structural adaptations can drive ubiquitination activity. Together, these findings provide a strong foundation to scale up and expand our studies, enabling deeper exploration of structure-function relationships as well as interactome networks to reveal novel regulatory pathways and mechanisms. This project seeks to decode how U-box and RING ligases integrate into and regulate broader cellular processes. It aims to uncover the molecular mechanisms of substrate specificity, ubiquitin transfer dynamics, and regulatory networks of these ligases. Leveraging structural and functional approaches in vitro and in planta, this research will elucidate plant stress responses and reveal conserved ubiquitination pathways with broad implications for eukaryotic biology and therapeutic innovation.

NIGMS - National Institute of General Medical Sciences

Project Summary The fusion of large dense-core vesicles (LDCVs) with the plasma membrane is essential for the secretion of peptides, hormones, and growth factors in neurons and endocrine cells. This tightly regulated process governs critical physiological functions, including metabolic homeostasis, immune responses, synaptic transmission, appetite regulation, circadian rhythms, and stress management. Disruptions in vesicle fusion are linked to neurological disorders, diabetes, immune dysfunction, eating disorders, depression, addiction, and cancer. Despite its significance, the molecular mechanisms of LDCV release remain poorly understood. The central hypothesis of this proposal is that distinct v-SNARE-containing vesicle populations form unique SNARE configurations, driving different modes of release and fusion pore kinetics. This innovative hypothesis challenges the prevailing view of a single core protein machinery governing vesicle fusion and suggests multiple vesicle types with varying calcium sensitivities. The unresolved debate between full fusion and the kiss-and-run (KR) mechanism has persisted due to technological limitations and the transient nature of these events. Our SLIM technology overcomes these barriers, enabling real-time tracking of vesicle fates and providing insights into the mechanisms behind KR and full fusion. This project will explore vesicle heterogeneity, synchronous and asynchronous exocytosis, and fusion pore kinetics, providing new insights into vesicle release mechanisms. Utilizing advanced technologies and cellular assays, this MIRA research program will address how vesicle heterogeneity dictates different fusion kinetics in LDCVs (Theme 1) and investigate the role of phospholipase in calcium signaling and lipid dynamics at fusion sites, while developing next-generation suspended lipid membrane platforms based on the design principles of organic neuromorphic devices for observing ultrafast fusion kinetics (Theme 2). The technologies we develop in this proposal will benefit the fields of membrane biophysics, cell biology, and single-molecule imaging. Ultimately, uncovering the principles of LDCV release will link intracellular and intercellular signaling with implications for treating metabolic diseases, psychological disorders, and neurodegenerative diseases. The results will impact numerous scientific fields, including cancer, cardiology, metabolism, and neuroscience.

NIDA - National Institute on Drug Abuse

Project Summary The widespread prescription and illicit use of opioids over the past several decades has created a public health epidemic known as the "opioid crisis." Synthetic opioids acting as Mu Opioid receptor (µOR) agonists have demonstrated effectiveness as pain management therapeutics yet have high addiction and overdose potential in addition to fatal side effects which have driven this ongoing crisis. Towards mitigating this crisis, naloxone is the primary therapeutic used to reverse opioid overdose, yet its use can precipitate withdrawal symptoms. Kappa Opioid receptor (κOR) has emerged as a promising target within the opioid receptor family, as selective antagonists have shown to counter emotional states and behaviors associated with withdrawal in preclinical studies. However, the utilization of κOR-targeted therapeutics has been limited by gaps in our understanding of receptor signaling and inhibition, resulting in unintended effects and unsuccessful clinical trials. As such allosteric modulators have gained attention due to their potential to provide a more fine-tuned way to modulate κOR signaling. As allosteric modulators occupy binding pockets that are distinct from the highly conserved orthosteric site, negative allosteric modulators (NAMs) can be drastically more selective than conventional antagonists. Furthermore, NAMs, by definition, enable residual endogenous signaling, which significantly broadens their therapeutic window. Until recently, no κOR-selective NAMs have been characterized. This proposal seeks to address these gaps by investigating the molecular mechanisms of a recently discovered NAM at κOR. By elucidating the structural and functional basis of κOR modulation via negative allosteric modulation, this research will contribute to the framework towards enhancing the clinical utility of κOR antagonists. To explore this fundamental aspect of κOR inhibition, we will delve into the molecular aspects of negative allosteric modulators (NAMs) in the context of our recent findings. Our first ever cryoEM structures of a novel conformational state of an inhibitor bound receptor κOR: G protein complex with inverse agonists JDTic, GB18 and norBNI highlight that inhibition is driven by highly complex mechanisms including the allosteric modulation of receptor-G protein affinities or even G protein nucleotide exchange which can result from stabilizing different states along the receptor activation pathway. Similar to orthosteric antagonists, NAMs may also stabilize distinct receptor states along the receptor activation pathway, promoting the inhibitory effects of orthosteric ligands. Taken together, this highlights the importance of investigating novel conformational states to provide a more complete picture of opioid receptor signaling and pharmacology. Using cryo-electron microscopy (cryoEM), in vitro, and ex vivo techniques to investigate inhibition via a κOR-selective NAM we will test the hypothesis that signaling inhibition arises from structural alterations that modulate G-protein dynamics. Through an extensive 1) Pharmacological Characterization of a κOR-selective NAM and 2) Structural Analysis of NAM-Induced Conformational States and Their Impact on Downstream Signaling we will link NAM-induced conformational changes at the atomic level to distinct pharmacological profiles. The results of this study will support the development of more effective therapeutic strategies at κOR and across the opioid receptor family.

NIA - National Institute on Aging

ABSTRACT RNA modifications represent a critical but poorly understood mechanism regulating protein production in aging muscle tissue. This proposal develops innovative computational and experimental approaches to map and understand how chemical modifications of RNA molecules change during muscle aging. Using a combination of cutting-edge nanopore sequencing technology and artificial intelligence, we will create new methods to precisely detect RNA modifications, particularly 5-methylcytosine (m5C), in muscle tissue. We will then apply these methods to examine how modification patterns differ between fast- and slow-twitch muscle fibers across age and sex, while determining their impact on protein synthesis. The project introduces novel synthetic RNA training strategies for improved modification detection while integrating chemical biology, machine learning, and muscle physiology. This work will establish new paradigms for studying RNA modifications in aging tissues while revealing fundamental mechanisms of muscle aging. The findings will identify potential RNA-based therapeutic targets for age-related muscle loss while providing valuable resources for the broader aging research community.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Aggression, a fundamental behavior essential for survival and species propagation, is ubiquitous across the animal kingdom. However, the neural underpinnings of innate aggression, particularly in terms of sexual dimorphism, remain inadequately elucidated. It is noteworthy that dysregulated aggression often surfaces as a symptom in various neurological and psychiatric disorders, with a pronounced prevalence in disorders more common among women, thereby underscoring a crucial gap in our scientific understanding. In response to this challenge, our research is strategically focused on the female Drosophila melanogaster to decipher the sexually dimorphic neural substrates governing aggression. Leveraging our prior breakthroughs in identifying neurons responsible for female-specific aggression, we have significantly enhanced the understanding of the mechanisms encoding sex-specific primal behaviors. In the next five years, we plan to employ the fly connectome for an extensive mapping of neural circuits, aiming to elucidate how stress influences aggression via evolutionarily conserved neuropeptides and to thoroughly investigate the developmental processes of these neural circuits. Moreover, our research will delve into how these circuits are disrupted in disease states associated with aggression dysfunction. Central to our efforts is the investigation of the modulatory effects of stress and peptides on female aggression and the assessment of the consequences of genetic modifications on key functional genes. Our systematic approach to dissecting the circuitry underpinning female aggression and identifying genes related to aberrant behavior is expected to yield profound insights into the genesis of sex differences in neural mechanisms that orchestrate social behaviors. This research program is poised to substantially advance our understanding of sex differences in brain functions related to the regulation of aggression across diverse species.

NIAID - National Institute of Allergy and Infectious Diseases

Project Abstract Allergic diseases result from dysregulated Type-2 immune responses to environmental allergens, with sensory neurons playing a key role in both allergen detection and immune activation. Sensory neurons, specifically TRPV1+ peptidergic (PEP) neurons, detect protease allergens, triggering itch and releasing Substance P, which activates dendritic cells to initiate Type-2 immunity. Recent findings reveal a bidirectional neuroimmune circuit involving sensory neurons and a subset of epidermal  T cells, termed GD3s, which produce IL-3. GD3s reside near free nerve endings, where their IL-3 primes Il3ra-expressing PEP1 neurons to enhance allergen detection and promote allergic immunity. Our long-term goal is to elucidate the mechanisms that regulate GD3 maintenance and function in both model organisms and humans. Type-2 immunity depends on feedback loops between sensory and immune cells that amplify allergic inflammation. Our central hypothesis is that sensory neurons not only respond to IL-3, but also sustain murine GD3s and their human counterparts – epidermal  T cells – through ligand-receptor interactions. Supporting this, we find that PEP1 neurons express Ccl8 and Bdnf, while GD3s express the corresponding receptors Ccr8 and Ntrk2, respectively. GD3s depend on Ccr8 for localization and proliferate in response to BDNF, suggesting that neurons actively regulate GD3 positioning and function. Furthermore, we find that human epidermal  T cells share common transcriptional signatures with murine GD3s, suggesting that epidermal  T cells may serve as the human correlate of GD3s. To test our central hypothesis, we will integrate cellular immunology, neurobiology, and molecular biology approaches across three specific aims: (1) Evaluate the chemokine-mediated pathways driving cutaneous GD3 localization, (2) Determine the neuronal factors driving GD3 function, and (3) Identify the human equivalent of GD3 cells. These studies will establish a fundamental understanding of the neuroimmune interactions that drive allergic immune activation, laying the foundation for therapeutic strategies to prevent allergic sensitization, treat chronic itch, and target chronic allergic inflammatory disorders.

NCI - National Cancer Institute

Project Summary/Abstract The emerging roles of granzyme K (GZMK)+ CD8+ T cells in conditions associated with defects in immune regulation, from rheumatoid arthritis to inflammaging, have garnered significant interest. The infiltration of GZMK+ CD8+ T cells is a prominent feature of the tumor microenvironment (TME) across several human cancers, and yet their role and regulation remain undefined. Recent studies indicate that tumor-infiltrating GZMK+ CD8+ T cells display distinct spatial and functional characteristics compared to other CD8+ T cells within tumors. Notably, high levels of GZMK+ CD8+ T cell infiltration are associated with poorer prognosis in certain cancers. Unlike other well- studied members of the granzyme family such as granzyme B (GZMB), the mechanisms driving GZMK expression in CD8+ T cells within the TME and the effects of CD8+ T cell-derived GZMK on antitumor immunity remain unclear. CD8+ T cell differentiation in the TME is shaped by the strength of T cell receptor (TCR) signaling upon interaction with tumor-specific antigen. Studies have shown that persistent and overly-strong TCR interactions drive T cell dysfunction, while insufficient TCR activation leads to functional inertness. Surprisingly, my preliminary data suggests that both these extremes of TCR signaling strength predispose CD8+ T cells to express GZMK. Furthermore, our group has demonstrated that GZMK can act as a novel initiator of the complement cascade, a known impediment to antitumor immunity through its ability to recruit suppressive myeloid cells to the TME. Building on these findings, I propose to investigate the mechanisms underlying GZMK expression in CD8+ T cells within the TME and its impact on antitumor immunity. I hypothesize that GZMK expression is driven in a subset of tumor-infiltrating CD8+ T cells that experience distinct TCR signaling, leading to the upregulation of context-specific transcription factors (TFs), and that eliminating GZMK from the TME will enhance antitumor immunity. To address my central hypothesis, in Aim 1, I will examine the impact of TCR signaling strength and cell-extrinsic signals on Gzmk expression in tumor-specific CD8+ T cells. First, I will assess the endogenous polyclonal CD8+ T cell response to cancer using Nur77 TCR-signaling reporter mice. Then, using OT-I CD8+ T cells and tumors with OVA-SIINFEKL variations, I will assess how controlled variation of TCR signaling strength impacts Gzmk expression. In Aim 2, I will identify the cis-regulatory elements and TFs that modulate Gzmk levels in tumor-infiltrating CD8+ T cells by perturbing key epigenetic and transcriptional regulators in vivo. In Aim 3, I will use Gzmk-deficient mice and CD8+ T cell-specific Gzmk-deficient mice to explore its impact on overall antitumor immunity. Together, these studies aim to provide critical insights into the regulation and function of GZMK in tumor-infiltrating CD8+ T cells and explore the potential of GZMK as a novel target for therapeutic intervention in cancer immunotherapy.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY Proper skin restoration after the damage is vital for organismal survival. The lymphatic vascular system is spread throughout the human body and has a critical function in mammalian physiology. In physiological conditions, its main function is the regulation of tissue drainage, immunosurveillance, and regeneration. Lymphatic dysfunction causes lymphedema a medical condition that manifests as tissue swelling and fibrosis. This serious condition results in profound severe delays in wound repair and the formation of chronic non-healing wounds. The mechanism by which lymphatic vessels regulate skin repair is unexplored. Additionally, while the role of lymphatic vessels in immune cell egress from tissues is well-established, how lymphatic vessels may directly modulate immune function in damaged tissues is poorly-defined. Recently, I discovered that lymphatic vessels are actively remodeled during wound healing to form small capillaries which are present in close localization to the wound front and hair follicles. This remodeling is critical for optimal repair as skin-specific loss of lymphatic vessels results in a significant delay in wound closure accompanied by a massive infiltration of immune cells. This proposal aims to leverage these observations by 1) delineating the mechanisms and consequences of lymphatic vessel remodeling during skin repair, and 2) determining the role of lymphatic vessels and fluid pressure in macrophage behavior during skin repair. This research stands to have a significant clinical impact because it can serve as a basis for developing new therapeutic avenues for lymphedema ulcers and chronic wound management. In addition, career-oriented guidance from my mentor and advisors, along with career development activated during the K99 phase that includes formal coursework on grant writing and project management, will further facilitate my transition to the R00 phase and my long-term productivity as an independent academic investigator. Collectively, the proposed research and career development plans are expected to generate data with a significant impact on understanding the repair and immunomodulatory functions of lymphatic vessels in skin repair and setting the basis of my future research as an independent researcher.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Establishing and maintaining immune tolerance to self-antigens is vital to prevent destructive autoimmune inflammation. Within the thymus, Aire plays a critical role in establishing self-tolerance, acting within medullary thymic epithelial cells (mTECs) to promote ectopic expression of tissue-restricted antigens which in turn leads to deletion of self-reactive T cells and generation of regulatory T (Treg) cells. However, a significant proportion of self-reactive T cells escape deletion in the thymus. A central unresolved question is how these self-reactive T cells are restrained within the periphery. To this end, we recently uncovered a novel lineage of antigen- presenting cells, named Thetis cells (TC), comprising four distinct subsets (TC I-IV). Our work thus far has demonstrated an essential role for TCs in extra-thymic Treg generation and tolerance to commensal and dietary antigens within the intestine. These studies established the tolerogenic potential of TCs. Whether and how TCs also regulate peripheral tolerance to self-antigens remain open questions. In a remarkable parallel, we found that TC I and Aire+ mTECs share a core transcriptional program that includes Aire and are critically dependent on the same transcription factors. These findings raise the tantalizing possibility that Aire+ TCs and Aire+ mTECs have shared immune regulatory functions. We hypothesize that Aire+ TCs restrain self-reactive T cells within the periphery through the expression of tissue-restricted self-antigens and immune-regulatory molecules. In the proposed project, we will determine i) the role of Aire+ TCs and extra-thymic Aire in peripheral immune tolerance, and ii) the molecular mechanisms by which Aire+ TCs mediate immune tolerance. Our preliminary data has established symmetry between Aire+ mTECs and Aire+ TCs and demonstrated the power of parallel analyses of these two cell types. In each aim, we will use novel genetic models and advanced computational approaches to address the Aire-dependent and independent functions of mTECs and TCs. In Aim 1 we will determine the role of Aire+ TC I in peripheral self-tolerance during steady- state and upon inflammation. In aims 2 and 3, we will address the mechanisms by which Aire+ TC I and mTECs regulate T cell tolerance, testing two non-mutually exclusive scenarios: expression of a ‘tolerogenic’ program that endows both Aire+ mTECs and TC I with the ability to induce T cell tolerance upon antigen presentation (Aim 2), and Aire-dependent regulation of tissue-restricted self-antigen expression (Aim 3). We anticipate that these studies will reveal fundamental mechanisms of immune tolerance and establish a new framework for self-tolerance. Elucidation of the role of TCs in peripheral immune tolerance will pave the way towards therapeutic manipulation of TCs with relevance to a broad range of autoimmune and inflammatory diseases.

NHLBI - National Heart Lung and Blood Institute

Project Abstract The alveolar epithelium is composed of two distinct cell types—alveolar epithelial type I (AT1) cells, which facilitate gas exchange, and alveolar epithelial type II (AT2) cells, which act as progenitors for AT1 cells. Successful lung repair following alveolar injuries requires AT2 cell proliferation and differentiation into AT1 cells, a process that involves the restructuring of gene regulatory networks and cell-type specific chromatin landscapes that underly these two cell fates. A dysfunctional regenerative response has been observed in a variety of severe lung diseases, involving AT2 cells acquiring a pathologic, alveolar-basal intermediate (ABI) cell state at the expense of an AT1 fate. The mechanisms that facilitate the cell fate decisions involved in AT2 cell maintenance and differentiation are not well understood, which has resulted in a lack of effective treatments to promote alveolar regeneration. This project aims to identify and characterize regulatory, 3-dimensional “hubs” of chromatin interaction that instruct distinct alveolar epithelial cell fates, and to determine how these hubs and their associated transcription factors regulate the acquisition of healthy and disease-associated states. Using human induced pluripotent stem cell (iPSC) models of AT1- and AT2-like cells (iAT1s and iAT2s), we will apply advanced chromatin mapping techniques to identify cell-type specific enhancer-promoter interactions and to characterize chromatin hubs that potentially regulate normal AT1 and AT2 cell identity. In Aim 1, we will map these interactions in healthy iAT1 and iAT2 cells, comparing their chromatin landscapes to pinpoint regulatory hubs that we hypothesize are responsible for cell-type specific gene expression. In Aim 2, we will explore the effects of haploinsufficiency of the lung lineage transcription factor, NKX2-1, on chromatin topology of iAT2 cells, hypothesizing that reduced NKX2-1 expression disrupts normal AT2 cell identity and favors a pathological ABI state. The findings from this research will enhance our understanding of the chromatin-based mechanisms that control lung cell fate decisions and provide insights into how disruptions of chromatin organization contribute to pulmonary disease.

NIGMS - National Institute of General Medical Sciences

Proteins are critical to human health and defects in them produce innumerable diseases. How thermodynamics is manifested, at a molecular level, in the various properties of proteins is central to maximizing our understanding of biology and deriving an ability to intervene and resolve human disease. Yet, despite amazing progress in biochemistry, biophysics and structural biology, the nature and role of entropy in establishing protein structure-function relationships remains remarkably obscure. Incomplete knowledge of protein entropy, a basic component of the Gibbs free energy that controls most aspects of molecular biology, is an unacceptable deficiency. Our long-term goal is to remedy that deficiency. Here we focus on the nature and role of protein conformational entropy (DSconf) in the thermodynamics of protein function. In a long-term effort, we have developed robust NMR-based strategies to probe DSconf. We have discovered an unanticipated role for DSconf in molecular recognition by proteins. With the necessary tools now in hand and having revealed this foundation, we propose to determine the role of DSconf in critical biochemical arenas: allosteric regulation; membrane protein stability and function; and the enzyme catalytic transition state. One pathway to disease comes from dysfunction of allosteric regulation of proteins. Allostery remains incompletely understood. Though the classical treatments have evolved to an ensemble-based view, the molecular details of the thermodynamics underlying allosteric transitions are unacceptably incomplete. Our hypothesis is that many instances of allosteric regulation are critically influenced by DSconf. As a model system, we will dissect the underpinnings of allostery in the E3 ubiquitin ligase Parkin, mutants of which cause early onset Parkinson's disease. Our work will open new views and provide a basis for intervention. These studies will also promote investigation of other E3 ubiquitin ligases, defects of which give rise to a large array of diseases. What we know from NMR-based studies of protein internal motion, and the DSconf that motion represents, has largely been derived from soluble proteins. We have recently overcome potent barriers to applying our approach to integral membrane proteins (IMPs). The first examples strongly indicate that IMPs are dynamically distinct from their water-soluble counterparts. This raises profound questions about how conformational entropy is accommodated and used in IMPs. We will gain unprecedented insight by examination of a set of well-positioned IMPs with important biological functions such as ion transport, signaling and enzymatic activity. Finally, it has long been a mystery about how the bulk of the protein supports the transition state during enzyme catalysis. Using the serine proteases as a test bed, we will challenge the hypothesis that changes in conformational entropy influence the energetics of transiting from the ground state Michaelis complex to the transition state. In summary, this proposal is unified by a focus on conformational entropy and will advance our understanding of the thermodynamic underpinning of protein function in a range of biomedically important contexts.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Metabolic disorders represent one of the largest burdens in our society, with an estimated 10-30% of adults affected by metabolic syndrome and over 11% of Americans diagnosed with diabetes. While genes that contribute to feeding and metabolism are well described, these phenotypes are highly polygenic, and the influence of individual alleles is shaped by genetic architecture. As such, a critical gap exists in our understanding of how naturally occurring genetic variation shapes feeding behavior and metabolism. Evolutionary medicine offers a powerful approach to explore how naturally occurring genetic variants impact feeding states, and the role genetic architecture plays in shaping these alleles. Evolution provides natural experiments that shape phenotypes in unique ways, and understanding how these phenotypes arise offers insights into how they change across taxa, including humans. We use the blind Mexican cavefish, Astyanax mexicanus, as a model to understand how naturally occurring genetic variation shapes feeding and metabolism. Astyanax exists as a single species in two forms: multiple populations of eyed, surface-dwelling fish and at least 30 populations of blind cavefish. We have shown that cavefish are hyperphagic, perpetually insulin-resistant (akin to diabetic), and exhibit slowed metabolism. We have also identified naturally occurring mutations in two well-known feeding receptors: melanocortin 3 (MC3R) and melanocortin 4 (MC4R). While the role of MC4R is well understood, much less is known about MC3R, and very little is known about how natural variations in these two receptors shape feeding. This proposal leverages innovative approaches developed in my lab over the past seven years to address this problem from a multidimensional perspective. Aim 1 will explore how MC3R and MC4R work together to regulate feeding and the impact of genomic architecture on these alleles. Aim 2 will use CRISPR-Cas9 to swap MC3R and MC4R alleles between surface fish and cavefish, allowing us to examine the impact on feeding behavior and neural activity in feeding centers. Aim 3 will assess brain-wide development in cavefish and explore the role of MC3R and MC4R in these processes. This proposal is innovative in its use of CRISPR-based allele swapping and advanced brain imaging techniques to uncover the genetic and neural mechanisms underlying these phenotypic variations. The overarching goal is to bridge the gap between laboratory-generated mutations and the complexities of natural genetic diversity, providing insights that could inform our understanding of obesity and metabolic disorders in humans.

St. Andrew’s Episcopal Church Foundation of Prineville

Decommission and Cleanup of UST

Jackson Creek Water Association, Inc.

Decommission Unapproved Well

NCI - National Cancer Institute

Summary Aneuploidy—the presence of chromosome gains and losses—is very rare in normal tissues but occurs in more than 80% of human tumors, especially in solid tumors. A high level of aneuploidy in tumors correlates with higher-grade disease, tumor progression, and resistance to therapy. Whether and how aneuploidy contributes to formation and progression of human tumors is not well understood. Whereas genomic and clinical studies in cancer patients suggest that aneuploidy drives tumorigenesis, experimental studies in mouse models and in vitro systems has so far yielded conflicting data on the role of aneuploidy in tumors. Human tumors are often specifically associated with either increases or decreases in the number of specific chromosome(s). One of the main obstacles to progress has been the technical limitation of not being able to engineer the ‘right’ type of aneuploidy in the ‘right’ cell type. Our ultimate goal is to dissect whether and how aneuploidy contributes to initiation and progression or human tumors. Our first objective here is to generate cellular models that faithfully recapitulate the aneuploidy patterns found in tumors in order to study how aneuploidy affects the pathobiology of tumor cells (their ability to grow in vitro or in vivo, to evade cell death pathways, to survive cellular stress and to regulate transcription and translation). Our second objective is to uncover vulnerabilities and synthetic lethal interactions potentially associated with the aneuploid state. The outcomes of the proposed project will represent the foundation to achieve the long-term goal of our lab, which is to develop a better understanding of the causes and consequences of aneuploidy in human tumors in order to uncover aneuploidy-associated biomarkers and therapeutic targets. To accomplish this goal, we will first use a panel of newly generated human cells containing different degrees and types of aneuploidy to compare diploid and aneuploid cells for several tumor-related cellular phenotypes both in vitro and in vivo. Secondly, we will adopt a systematic approach to identify genes and pathways that when blocked, inhibit proliferation and survival of aneuploid tumor cells but not normal cells. Third, we will perform a protein and phospho-protein analysis mainly of colorectal tumor patients’ samples to dissect the consequences of aneuploidy at the level of protein stability and pathway regulation. Our results will fill an important gap of knowledge in our current understanding of how aneuploidy evolves during tumorigenesis and how we might take advantage of this knowledge to improve patient outcomes.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Psoriasis (Ps) is an autoimmune-mediated systemic inflammatory skin disease. Approximately one-third of Ps patients eventually develop psoriatic arthritis (PsA). PsA is one of the most common forms of arthritis, impacting around 1 million individuals in the US. Mechanical pain associated with PsA in joints and skin, triggered by joint loading, movement, and skin touch, is a leading cause of hospitalization for patients and represents a serious unmet medical need. Current PsA therapeutics, including recently developed interleukin 17 (IL17), interleukin 23 (IL23), and Janus kinase (JAK) inhibitors, only offer partial relief from pain. Furthermore, these treatments prove ineffective for some individuals and have side effects. Therefore, there exists a pressing need to identify new targets for PsA pain. Animal pain models can facilitate the rational discovery of novel analgesics. Although current animal models of PsA have advanced our understanding of PsA pathogenesis, it is unknown if they can be used for studying PsA pain because pain has not been characterized in any of these models. Importantly, most of them have limitations that hinder their use for studying chronic PsA pain. We propose to improve, characterize, and validate a mouse mannan model for PsA pain (Aim. 1). Due to the lack of animal models, we know little about the mechanisms underlying PsA pain. Emerging evidence demonstrated that the systemic and local tissue levels of lysophosphatidylcholine (LPC), a proinflammatory mediator and the most abundant systemic phospholipid in humans, are elevated in psoriatic patients, suggesting its potential involvement in the pathogenesis of PsA pain. Using our improved and validated mouse model in Aim 1, we will examine whether elevated LPC contributes to PsA pain (Aim 2). Our previous work and preliminary data demonstrated that LPC can activate primary sensory neurons, joint chondrocytes, and skin keratinocytes via a mechanosensitive ion channel TRPV4. These findings prompted us to ask whether LPC drives PsA joint and cutaneous pain via TRPV4 in sensory neurons, chondrocytes, and keratinocytes (Aim 3). Success completion of this project will lead to the development of the first animal model of PsA pain that can be used to substantively advance our understanding of PsA pain mechanisms. In addition, this project will pave the way to identify LPC and TRPV4 as mechanistic therapeutic targets with exciting potential to mitigate PsA pain.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY The putative oncogene DDX6 (RCK, p54) has critical roles in the nervous system, embryonic cells, and somatic cells. The importance of DDX6 is further emphasized by rare pathogenic missense mutations in DDX6 that are associated with developmental delays and intellectual disability in children. DDX6 is a member of the DEAD-box RNA helicase family, which are broadly involved in regulation of messenger RNA (mRNA) and translation. Despite its establishment as critical regulator of translation initiation, the molecular mechanisms that underlie DDX6 function remain unclear. Major roadblocks to this include that translation initiation is a heterogenous, multistep process that requires a dozen components and occurs within a minute. Further, the identity of interacting partners in a given DDX6 regulatory complex determines whether DDX6 promotes or inhibits protein synthesis. This complicates cellular study of DDX6, as DDX6 likely forms multiple distinct regulatory complexes on different mRNAs at any given time. To overcome these roadblocks, I will combine biochemical, single-molecule, and biophysical methods to examine my central hypothesis that DDX6 regulates the initial, rate-limiting step of translation initiation and global mRNA conformation in concert with its interaction partners. In Aim 1, I will identify the step of translation initiation that is regulated by DDX6 and its key partner proteins by using in vitro single molecule fluorescence microscopy and a reconstituted human translation initiation system. In Aim 2, I will examine how DDX6 assembles with partner proteins into distinct regulatory complexes and how this impacts mRNA dynamics. In each Aim, I will determine how pathogenic missense mutations in DDX6 disrupt its function. My proposed experiments will uniquely leverage my background in protein biochemistry and enzymology, the Lapointe lab’s expertise in human translation initiation and single molecule fluorescence microscopy, and the Stoddard lab’s expertise in structural biology and enzymology. My collective findings should elucidate which step of translation initiation is controlled by DDX6, how DDX6 assembles into translation inhibiting and translation stimulating complexes, how distinct DDX6 complex assemblies remodel mRNA, and how missense mutations alter DDX6 function. In parallel, my individualized training plan will provide me with opportunities to expand my research skillset into single molecule techniques and human translation initiation, to grow as a leader and mentor within the Lapointe lab, and prepare me for a career as an independent investigator.

NIDA - National Institute on Drug Abuse

More than 50% of people living with HIV (PLWH) encounter cognitive dysfunction, and chronic peripheral pain, in the setting of opioid drug abuse. Neuronal circuitry innervating from the prefrontal cortex to the striatum is important in decision-making. Additionally, dopaminergic projections from the ventral tegmental area (VTA) in the midbrain to the nucleus accumbens (NAc) and dorsal striatum are involved in reward and motivational behaviors. We reported that global suppression of secreted phosphoprotein-1 production (OPN/Spp1) increases the expression of the mitochondrial translocator protein (TSPO) in Iba1+ macrophages/microglia across several key brain regions involved in cognition including the substantia nigra (SN). Interestingly, a subset of tyrosine hydroxylase (TH) reactive neurons co-labeled with TSPO, or were closely positioned near TSPO+ TH- cells in the midbrain region. In this regard, substance use disorders (SUD) in PLWH and in particular methamphetamine (METH) continues to be a consequential comorbid condition impacting viral suppression and health outcomes. Findings using rat and mouse HIV-Tat transgenic models have provided insights about how METH alters the expression of genes required for dopamine synthesis, metabolism, and receptor trafficking, and the role of sex as a modifier. However, our understanding of how dopaminergic neuronal-glial communication is altered in vivo during HIV replication, and METH is not understood likely due to the daunting task of deconvoluting multiple intersecting variables. Moreover, we implicated mammalian target of rapamycin (mTOR) pathway activation by (OPN/Spp1) in a mechanism of neuroprotection. Whether mTOR-OPN/Spp1 signaling plays a role in microglial- dopaminergic neuronal crosstalk in HIV-METH infection-exposure in vivo in is unknown. In this R21 application for high-risk/reward ideas, we propose to use our expertise with HIV-infected humanized mice, SUDs and modeling, to develop a rational approach to mathematical model dynamic dopaminergic neural-glial network circuitry. Our overarching hypothesis is that neuro-glial cells upregulate OPN/Spp1 expression in response to HIV-1 infection, which stimulates mTOR pathway signaling thereby, activating neuroprotective signaling to preserve homeostatic neurocircuitry; with acute co-exposure to METH, these pathways are upregulated and reinforced in a time-dependent manner. We will interrogate gene expression among neurons and glia in the nigrostriatal and meso-limbic brain regions to resolve dopaminergic neuronal-glial interactions using prospectively collected in vivo time course data. The analyses will focus on identifying interactions in the presence of HIV infection, with and without ART and acute administration of METH. Our long-term goal is to gain insights into the therapeutic potentials of resilience from cognitive dysfunction and drug addiction. We expect that findings from this project will advance the understanding of immunomodulation and metabolic reprogramming during co-exposure to HIV-1 and METH and will provide avenues for translational and clinical research aiming at improving mental health and substance use.

Agassiz Baldwin Community

DeCordova Museum & Sculpture Park

East End House

DeCordova Museum & Sculpture Park