University Of Texas At Austin

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY We will develop a data-driven model of seasonal and pandemic influenza transmission throughout the US to accelerate robust assessments of multifaceted influenza intervention strategies. We will work closely with the CDC Modeling Network to advance the fidelity, transparency and translation of models as an evidence base for influenza policy making, prevention and control. This project extends a metapopulation model of influenza transmission within and between 217 major metropolitan areas in the US that we are developing in collaboration with the CDC Modeling Network. The model includes travel between cities, age- and risk-group specific susceptibility, probability of clinical outcomes, intervention efficacies and uptake rates, as well as the impacts of local climate and school calendars on transmission rates. Using a range of public health, epidemiological, societal and economic metrics, the model can flexibly evaluate thousands of candidate intervention strategies, including time- and location-based combinations of vaccines, antivirals, and social distancing measures with potential subgroup-specific prioritization. Our proposal includes four major aims. In Aim 1, we will extend our US Influenza Model to include the co- circulation of multiple viruses competing via transient heterosubtypic immunity. We will derive new estimates for the duration and magnitude of heterosubtypic immunity and design strain-specific strategies for effectively controlling co-circulating seasonal and pandemic influenza viruses. In Aim 2, we will evaluate intervention strategies that leverage newly approved and combined antiviral drugs. We will fit within-host viral dynamic models to clinical data on new antivirals to estimate the efficacy of various drug regimens in different subpopulations with respect to disease severity, infectiousness, and the risk of antiviral resistance. In Aim 3, we will build a granular within-city model of influenza transmission based on abundant data and local collaborations with public health and healthcare leaders in the Austin-Round Rock Metropolitan Area. We will apply the model to elucidate socioeconomic and geographic disparities in influenza risk and design interventions that ameliorate such gaps. In Aim 4, we will build an interactive visualization platform that allows users to specify epidemic scenarios, implement layered interventions as simulations unfold, and view the model dynamics through the lens of a surveillance module based on the CDC’s FluView Interactive portal. We will work extensively with the CDC Modeling Network to build a diverse portfolio of validated models and best practices for collaborative decision support. Our projects will contribute flexible models for the evaluation of multifaceted influenza interventions, elucidate competition among influenza viruses and the efficacies of novel antivirals, and provide insights into socioeconomic disparities in influenza burden. Furthermore, our innovative visualization tool will broadly support the translation of science to public policy.

NIH Research Projects · FY 2025 · 2020-09

Project Summary / Abstract Cell polarity is a fundamental feature of eukaryotic cells, and must be coordinated between cells and regulated to allow for normal animal development and tissue homeostasis. Despite genetic identification of proteins involved in cell polarity and a large body of knowledge about their interactions in vitro, it remains unclear how polarity proteins are organized into signaling complexes in cells. This lack of knowledge has prevented the field from understanding mechanisms of developmental control of polarity signaling in vivo. The long-term goal of the proposed research is to resolve the network of protein-protein interactions that supports animal cell polarity and to understand how this network can respond to developmental signals. To enable progress towards this goal, the applicants have developed innovative experimental tools that allow single-molecule measurements of native protein complex abundance in single cells. This project focuses on an evolutionarily conserved protein kinase, called aPKC, that plays a central role in polarity by localizing to one end of a polarized cell and dictating polarized cell behaviors. The applicants will make use of the C. elegans early embryo, in which cells reproducibly polarize in response to multiple spatial and temporal cues, to discover mechanistic links between developmental signals and the polarity machinery. The central hypothesis of this work is that developmental signals control cell polarity by altering the molecular complexes in which aPKC resides. This hypothesis will be explored by elucidating mechanisms that regulate assembly of aPKC into different complexes in the zygote (Aim 1); by determining how polarity is entrained to cell-cell contacts in 8-cell embryos (Aim 2); and by determining how translation of new protein components remodels the polarity system between these two stages (Aim 3). The work proposed in this application is significant because it will reveal fundamental mechanisms controlling cell polarity, and because it places these mechanistic studies in a developmental context. The proposed work is innovative, in the applicant’s opinion, because it uses novel experimental methods to perform biochemical, mechanistic studies in vivo. By studying the biochemical control of aPKC in multiple cellular and developmental contexts in a single experimental system, this work will identify fundamental mechanisms of PAR polarity signaling and to learn how these mechanisms are deployed to achieve different outcomes during development.

NIH Research Projects · FY 2024 · 2020-08

This proposal presents a research career development program focused on the study of alcohol’s dose-dependent effects in neuroinflammation in a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). I am currently an assistant professor of neurology at Dell Medical School at UT Austin. The outlined proposal builds on my previous research in neurobiology of sex differences, clinical training in neuroimmunology and integrates new domains of expertise in bioinformatics, alcohol and microbiome research, and basic science neuroimmunology. I will be guided by outstanding mentors and advisors, including Drs. Adron Harris (alcohol research), Hans Hofmann (bioinformatics), Sergio Baranzini (microbiome), Olaf Stuve (neuroimmunology), and William Schwartz (career guidance). The proposed experiments, didactics, and mentorship will enable me to transition to an R01 funded physician scientist in the cross disciplinary field of neuroimmunology and alcohol research. MS is a chronic autoimmune demyelinating disease of the central nervous system (CNS) and the leading acquired cause of neurological disability in young adults. The cause of MS is unknown. Although genes contribute to the disease risk, it is thought that environmental factors, such as diet and the gut microbiome contribute to a larger degree of the risk. Alcohol is a common dietary factor used by MS patients. Yet, despite its widespread use, potential for abuse and known gut, CNS and immune effects, alcohol’s role in MS is not well understood. The foundation of this proposal is based on my preliminary studies, in press in PNAS, demonstrating that moderate alcohol consumption leads to EAE amelioration, decrease in microglia in the spinal cord, and a shift of gut microbiota toward a regulatory phenotype in a sex-specific pattern, that collectively suggest a protective role of moderate alcohol in EAE and potentially in MS. Given known pro-inflammatory effects of alcohol, these studies raise the question of alcohol’s possible differential effects on neuroinflammation at high vs moderate doses. This proposal begins to address this question by evaluating alcohol’s dose-dependent effects on the peripheral and CNS immune system and the gut microbiome. Specifically, the aims of this proposal are (1) What are the peripheral and CNS immune cell subsets driving dose-dependent alcohol effects in EAE? Can adoptive transfer from alcohol-consuming mice recapitulate clinical symptoms in naive mice? and (2) Which gut microbiome constituents are responsible for alcohol’s dose-dependent effects in EAE? Can microbiome transfer from alcohol-consuming mice recapitulate clinical symptoms in naive mice? The scientific objective of this proposal is to begin to define alcohol’s dose-dependent effects in neuroinflammation by examining the immune system and the gut microbiome with the vision of generating hypotheses that can inform the direction and design of future diet studies in EAE and MS and expand the repertoire of available and targeted probiotics for MS patients.

NIH Research Projects · FY 2024 · 2020-08

Project Summary/Abstract: This K01 research and training award would advance my long-term goal of devel-oping and evaluating culturally and developmentally appropriate Latino youth alcohol use preventive interven-tions. Compared to Blacks and Whites, Latinos in middle adolescence (ages 14–16) are disproportionately at risk for alcohol use. Although by late adolescence (ages 18-20) rates of underage drinking for Latinos is similar to those for Whites, alcohol use risks (e.g., drunk driving, unplanned/unprotected sex) are more severe for Lati-nos. Normative adolescent stressors partly explain why many youth initiate alcohol use. Yet, Latino youth and families face additional stressors related to prejudice, discrimination, and racism, leaving Latino youth vulnera-ble to higher alcohol use/related risks. Many studies have documented the impact of prejudice, discrimination, and racism on Latino youth alcohol use. Yet, we know almost nothing of how to prepare Latino youth and fami-lies to live in a world in which prejudice, discrimination, and racism are part of their daily life. Evidence-based preventive interventions for Latinos have not targeted coping skills for prejudice, discrimination, and racism. Yet, it might be precisely these skills that youth and parents are lacking that are driving Latino youth alcohol use disparities. Although, evidence-based preventive interventions for Latinos exist, Latino youth continue to be at disproportionately higher alcohol use risk, suggesting that more preventive intervention development work is needed. The overarching goal of my K01 research plan is to develop and pilot test a family-based youth alcohol use preventive intervention that will equip youth and parents with coping skills to manage preju-dice, discrimination, and racism. My training plan will provide me with the skills necessary to become an inde-pendent youth alcohol use prevention scientist. I will acquire training in qualitative/mixed methods used in inter-vention development; the development and implementation of preventive interventions; and the evaluation of preventive intervention outcome data. My training plan is directly linked with my three research aims. For Aim 1, I will use qualitative methods to inform the development of intervention sessions that target coping with prej-udice, discrimination, and racism. For Aim 2, I will create intervention sessions that target coping with preju-dice, discrimination, and racism. I will draw from strategies used in existing interventions and from Aim 1 focus group data. I will seek feedback from youth and families before finalizing the curriculum. For Aim 3, I will pilot test the intervention among Latino families; assess intervention feasibility and acceptability; and determine pre-liminary effect size estimates for the intervention’s promise to improve coping skills, family functioning, parent-ing behaviors, and youth alcohol outcomes. The proposed research will contribute to the first family-based La-tino youth alcohol use preventive intervention targeting coping skills for prejudice, discrimination, and racism. It will provide me with the training, experience, and pilot data necessary for a fully powered randomized trial (R01), facilitating the leap to independent investigator and Latino youth alcohol use prevention scientist.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY The overall goal of this K23 proposal is to provide Trisha Parekh, D.O., with the career development and research experience necessary to allow her to become an independent researcher in chronic obstructive pulmonary disease (COPD) health disparities, with expertise in social care integration and psychological stress reduction in low-income communities using community health worker interventions. Disadvantages of social determinants of health, such as low socioeconomic status, are associated with adverse outcomes in COPD, including more frequent hospitalizations, early readmissions, and increased mortality. A majority of interventions to reduce readmission focus on addressing medical determinants of health, with a notable absence of addressing social needs that impede better health outcomes. The psychological stress associated with poverty and other disadvantages of social determinants of health can lead to negative downstream behavioral, psychological, and physiological effects. For COPD patients, these stress responses may include increased tobacco use, increased risk of anxiety or depression, or increased systemic inflammation, all of which are risk factors for acute exacerbations of COPD. Substantial knowledge gaps on the social determinants of health, their associated levels of perceived stress in COPD, and clinical outcomes associated with high levels stress in marginalized groups must be addressed in order to reduce health disparities and improve quality of life for these individuals. In this K23 proposal, under the guidance of an expert mentorship team, Dr. Parekh will conduct a mixed methods research study to inform the development of a targeted community health worker-led stress reduction intervention in low-income COPD patients that will focus on 1) addressing unmet social needs, 2) providing COPD disease education, and 3) promoting proactive coping behaviors. The specific aims for this research project are as follows: Aim 1: Identify modifiable social determinant of health predictors and clinical outcomes of perceived stress in COPD patients; Aim 2: Understand perceived stressful experiences in high-risk COPD patients; Aim 3: Develop and test the feasibility of a community health worker intervention to reduce stress in low-income COPD patients. At the completion of Aim 3, this intervention will be ready to be tested in a R-series grant funded randomized controlled pilot with the primary outcome of stress reduction and secondary outcomes of reduction in acute care use and tobacco use and improvement in quality of life. At the conclusion of this K23, Dr. Parekh will have the training and experience needed to transition to a successful independent investigator who has an expertise in COPD health disparities.

NIH Research Projects · FY 2025 · 2020-07

ABSTRACT Manganese (Mn) is an essential metal, but elevated levels induce severe neurotoxicity that has no treatment. While the direct neurotoxic effects of Mn are well studied, Mn is predominantly excreted by the liver and intestines, and a high prevalence of neurotoxicity associated with elevated blood and brain Mn levels is reported in patients with liver disease. Moreover, recent epidemiological studies suggest that alterations in Mn excretory capacity are widely prevalent in the general population due to common genetic polymorphisms. Yet, the role of Mn excretion in modulating the outcomes of Mn induced neurological disease is unclear, and the critical question of whether the risk of Mn neurotoxicity depends on Mn excretion capacity has not been answered. Until recently, a major limitation in studying the relationship between Mn excretion and neurotoxicity was that the mechanisms of Mn excretion were unknown. Our recent work, supported by a NIEHS “ONES” R01, revealed that the combined activities of two Mn transporters, SLC30A10 and SLC39A14, were necessary for Mn excretion — SLC39A14 transported Mn from blood into the liver and intestines, and SLC30A10 excreted the intracellular Mn into bile and feces. Further, our analyses of Slc30a10 and/or Slc39a14 knockout mice demonstrated that brain Mn levels were primarily regulated by the excretory activities of these transporters in the liver and intestines. Based on the predominant role of excretion in controlling Mn levels in the brain, we hypothesize that hepatic and intestinal Mn excretion are critical modulators of the risks and outcomes of Mn neurotoxicity. We will test this hypothesis by leveraging liver or intestine specific Slc30a10 knockout or knockin mice, which we generated in the ONES phase, as novel human relevant models to decrease or increase Mn excretion, respectively. Aims 1 & 2 will directly establish the regulatory role of hepatic and intestinal Mn excretion in Mn neurotoxicity. In Aim 1, we will determine whether liver or intestine specific Slc30a10 knockout mice exhibit heightened sensitivity to Mn neurotoxicity. In Aim 2, we will test whether liver or intestine specific Slc30a10 knockin mice are protected against Mn neurotoxicity. Studies in Aim 2 have high translational relevance as they may identify increasing Mn excretion to be an effective strategy for the management of Mn neurotoxicity. Aim 3 will build on our finding that elevated Mn exposure enhanced SLC30A10 expression via Hif1α in the liver, providing a means to increase Mn excretion during toxicity. In Aim 3, we will elucidate the mechanisms and determine whether this response protects against neurotoxicity. These studies will provide foundational information about protective responses to Mn at the organism level, which have not yet been described, and set the stage to test if Hif1α activators, in clinical trials for other diseases, can be repurposed for Mn neurotoxicity. In sum, our studies will provide fundamental insights into a central, but overlooked, aspect of Mn neurotoxicity, and aid in the development of novel therapeutic approaches for Mn induced neurological disease.

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY/ABSTRACT The proposed research involves continued studies of group II intron and related reverse transcriptases (RTs), their biological functions, biochemical mechanisms, and RNA-seq applications, including analysis of clinical samples for RNA-diagnostics and liquid biopsies. Group II intron RTs are encoded by mobile group II introns, bacterial and eukaryotic organellar retroelements that were the evolutionary ancestors of introns, the RNA splicing apparatus, and retroelements in humans. They differ structurally and functionally from retroviral RTs and have biochemical activities advantageous for RNA-seq and genome engineering applications. Group II intron RTs belong to a larger RT family termed non-LTR-retroelement RTs, which includes human LINE-1 and domesticated bacterial RTs that evolved from group II intron RTs to perform cellular functions. Previously, we developed general methods for purifying group II intron and related RTs with high yield and activity and applied them to group II intron RTs from bacterial thermophiles to obtain Thermostable Group II Intron RTs (TGIRTs). We obtained an X-ray crystal structure of a full-length TGIRT in complex with template-primer and incoming dNTP, a first for any non-LTR-retroelement RT, and used TGIRTs to develop an advantageous high-through- put RNA-seq method (TGIRT-seq) for comprehensive profiling of protein-coding and non-coding RNAs in hu- man cells, extracellular vesicles (EVs), and plasma. During the current grant period, we worked out mecha- nisms used by a related bacterial RT-Cas1 fusion protein to site-specifically integrate RNA spacers into DNA genomes; found that another domesticated bacterial group II-like RT (G2L4 RT) evolved to function in double- strand break repair (DSBR) by microhomology-mediated end joining (MMEJ); used TGIRT-seq to characterize a large novel class of short structured full-length excised linear intron RNAs (FLEXIs) in human cells and plasma; found that FLEXIs and other classes of introns with binding sites for the same subsets of RNA-binding proteins (RBPs) were enriched in functionally related host genes, whose expression might thus be coordinately regulated by these RBPs; developed TGIRT-seq methods for parallel analysis of transcriptional and post-tran- scriptional gene regulation; and used these methods to analyze inflammatory breast cancer (IBC) clinical sam- ples, leading to new insights into IBC and the identification of potential IBC biomarkers in cells and plasma. In the proposed research, we will investigate: (i) the structural basis and physiological significance of novel mech- anisms used for initiation of cDNA synthesis by an RT-Cas1 protein shared by bacterial group II intron and hu- man LINE-1 RTs; (ii) the mechanism and structural basis for DSBR by G2L4 RT, extending these studies to group II intron and LINE-1 RTs for which we found DSBR via MMEJ is an inherent activity; (iii) explore the in- volvement of LINE-1 elements in DSBR in human cells; (iv) use TGIRT-seq to analyze cancers; and (v) investi- gate the physiological significance and mechanisms that lead to enrichment of FLEXIs and intron RNA frag- ments in EVs and plasma, including a class of FLEXIs that may function in gene regulation.

NIH Research Projects · FY 2025 · 2019-09

Craniofacial dysmorphologies are some of the most common human birth defects and can be extremely variable in their severity and extent. Understanding, diagnosing and treating these dysmorphologies requires a detailed understanding of the normal genetic hierarchies, cell signaling and cellular interactions that drive the morphogenesis and integration of the multiple cell types within the craniofacial complex. Genetic and/or environmental perturbations that disrupt these normal processes can cause craniofacial dysmorphologies. Given that human craniofacial birth defects tend to be sporadic and non-syndromic, it is most likely that multifactorial perturbations are the most common cause of these defects. However, there are major gaps in our understanding of the normal processes in craniofacial development. Furthermore, we have very little understanding of how multifactorial interactions disrupt normal development. This R35 proposal will support the unique research program of Dr. Johann Eberhart’s lab. The signaling interactions mediating craniofacial morphogenesis are examined in the first program. The second program examines how craniofacial tissues integrate seamlessly with one another. The third program examines the environmental and gene-environment interactions that can disrupt craniofacial development. Dr. Eberhart is an extremely well trained and productive developmental biologist. He has been supported through various NIH-based mechanisms ever since he was an undergraduate student. He has authored 40 total publications, 28 since establishing his independent lab at UT Austin. He has established himself as a leader in the field of craniofacial development and has pioneering publications on muscle-tendon attachments, zebrafish palatal development and gene-environment interactions. Dr. Eberhart is routinely invited to present the work from his lab at national and international meetings. He is also regularly asked to provide scientific service, such as grant reviews and organizing/participating in scientific workshops. Dr. Eberhart is a tireless mentor. He not only ensures that his trainees research is top notch, but is also deeply engaged with their scientific careers. To date, every one of Dr. Eberhart’s trainees that have been eligible for fellowships have received at least one. Dr. Eberhart is also committed to increasing diversity in STEM fields and actively recruits promising young scientists from diverse backgrounds into the field.

NIH Research Projects · FY 2025 · 2019-09

Project Summary/Abstract Metals are essential micronutrients that are required for proper functioning of cells and organisms, and their distribution and speciation (the metallome) is tightly controlled by complex homeostatic machinery. Deviations from metal homeostasis are associated with multiple pathologies, environmental metal contamination, and metal deficiencies, which all have important effects on cellular function. The metallome is defined by two main metal ion pools: the labile metal ion pool in which metals are weakly bound and exchangeable and the tightly-bound metal ion pool in which metals are ligated to biomolecules with high affinity. Metalloproteins that constitute the tightly-bound metal ion pool represent one third of the proteome and within these proteins, metals have diverse structural and catalytic functions. These metalloproteins exist in several metalation states, including apo (metal- free), holo (metal-bound), and mismetalated. These states are controlled by several factors, including metal ion availability and the presence of metal transporters or metallochaperones. The Que lab is interested in studying how the metalation state of metalloproteins in the cellular environment responds to changes in metal ion availability and other biological stimuli. There are several broad questions that drive our research: (1) are there biological or abiological scenarios in which metals in metalloproteins are labile and exchangeable rather than tightly-bound, making these proteins vulnerable to metal ion loss or demetalation? (2) What factors govern how vulnerable a metalloprotein is to demetalation? (3) How does this knowledge impact our understanding of the biological function of these enzymes and their relation to human health and disease? Our strategy to tackle these questions is to develop fluorescent probes that allow us to monitor metalloprotein expression and metalation dynamics in live cells. We specifically target metalloenzymes due to the important chemical reactions they catalyze and the presence of an open coordination site in their active site that can be targeted using metal- binding, inhibitor-inspired fluorogenic molecules. In the previous granting period, we demonstrated our ability to produce fluorescence turn-on probes for the ubiquitously expressed carbonic anhydrase (CA) and antibiotic resistance enzyme New Delhi Metallo-𝛽-lactamase (NDM). These probes revealed that CA-bound zinc is not labile in cells whereas NDM-bound zinc is labile, with metal loss being observed after NDM-expressing E. coli were treated with metal chelators. In the next five years, our first goal is to improve the properties of our fluorescent probes to increase their sensitivity and enable multiplexed imaging. Our second goal is to target additional metalloenzymes in order to expand our biological scope and elaborate on our probe design principles. Our third goal is to further explore the lability of zinc sites in three clinically relevant metallo-𝛽-lactamases, including NDM, and the consequences of this lability on antibiotic resistant infections, the nutritional immunity response, and therapeutic strategies. Metalloenzyme probes developed as part of this award also have potential applications in diagnostics and high-throughput drug screening for pathologies associated with these species.

NIH Research Projects · FY 2026 · 2019-08

SUMMARY Hydrogen bonding in crowded environments – impact on biomolecules: Biomolecular organization in vivo is driven by crowding and heterogeneity. To date, protein structure, dynamics, and folding have been studied almost exclusively in buffer solutions, yet cellular environments are highly complex. This project seeks to characterize the driving forces behind protein structure and dynamics in environments that mimic the cytosol. Specifically: 1. We will quantify the sequence and crowder-dependent impact on the solvation shell of biomolecules, by mapping out the changes in solvent configurations and dynamics. 2. We will link changes in the microscopic H-bond environment with bulk effects on protein structure and stability. 3. We will advance 2D IR spectroscopic techniques to perform in-cell 2D IR measurements using labeled proteins in live mammalian cells to measure H-bond dynamics in the cytosol. We will directly address the question of “what are the properties of biological water?” which has been one of the key unresolved questions in the field. Liquid-liquid phase separation (LLPS) – biocondensate structure, dynamics, and interactions: Despite the popularity of biocondensates, the field has remained highly empirical. The residual secondary structure and stability of biocondensates, specifically the balance between electrostatic interactions and hydrogen bonding is not well understood. We seek to: 1. Investigate H-bond dynamics in partially disordered low-sequence- complexity condensates. We will induce LLPS using fusing Cryprochrome-2, a light-activated oligomerization protein to disordered domains (RGG) to generate “strong” (high physical crosslinking) and “weak” (low crosslinking) condensates in situ by UV illumination. We will probe the role of local interactions by comparing 2D IR measurements when LLPS is induced through crosslinking, crowding, or electrostatic interactions using RNA oligos. 2. We will investigate residual secondary structure in intrinsically disordered domains. Specifically, does LLPS promote folding. What is the role of LLPS in stabilizing secondary structure? Rational design of biologics – protein-polymer interactions: Proteins represent a rapidly emerging approach to therapeutics, though rapid degradation and low bioavailability pose a significant challenge. Polymer conjugation (PEGylation) is used to stabilize the proteins and increase bioavailability. However, most of the knowledge is empirical, as molecular models to explain how polymers interact with the protein surface are lacking. We will address the following hypotheses: 1. PEG-protein interactions are highly dynamic without specific “long-lived” contacts. Surface dehydration, or “water replacement” alters the backbone structure and dynamics. 2. Polymers modify thermal denaturation pathways. 3. PEG allergies highlight the need to explore alternative polymer architectures to avoid immunogenicity. Here, we will explore a series of alternative compositions including linear and branched polymers.

NIH Research Projects · FY 2024 · 2019-07

DESCRIPTION (provided by applicant): Prenatal alcohol exposure (PAE) is a leading cause of intellectual and other brain disabilities, contributing to an estimated prevalence of Fetal Alcohol Spectrum Disorder (FASD) at between 1 and 5% of school-aged children in the US. Despite prevention guidelines, alcohol use during pregnancy continues to be a problem, and consequently, FASD is difficult to prevent. Behind every child with an FASD is an adult with unmet mental health needs that result in risky patterns of alcohol consumption or an Alcohol Use Disorder (AUDs). Therefore, preventing FASD requires preventing risky alcohol consumption in adults. Preventing AUDs is challenging due to the paucity of effective medications. In this application, I propose a transitioning plan in which I complement my passion for the study of FASD (the F99 phase) with the study of AUDs (the K00 phase), with the expectation that the route to preventing FASD lies through preventing AUDs. However, in both phases, I plan to pursue my interests in the mediating biology of non-protein-coding RNAs. In the first phase of my predoctoral studies, I focused on Oct4/Pouf51, a transcription factor that is a key determinant of stem cell identity, and target of ethanol. I also identified a novel pseudo- gene duplication of the Oct4/Pou5f1 locus, encoding a long non-coding RNA that I termed, Oc4pg9 lncRNA. Oct4pg9 lncRNA is upregulated in neural stem cells (NSCs), following ethanol exposure. I found that Oct4pg9 lncRNA mediates many maturational effects of ethanol on NSCs. In the F99 phase, I plan to assess, using single-cell RNA sequencing, whether the expression of Oct4pg9 lncRNA and Oct4/Pou5f1 marks unique non-overlapping NSC subpopulations. Using an in vivo murine model for PAE, I plan to determine the extent to which ethanol exposure disrupts, at the cellular level, the association between Oct4Pou5f1 and Oct4pg9, leading to the emergence of new NSC subpopulations with aberrant maturation signatures. In the K00 phase, I will transition to the field of adult alcoholism and continue studying the regulation and function of lncRNAs in the context of AUDs. This research direction will focus on developing and behaviorally phenotyping mouse models of AUD-sensitive lncRNAs. Additionally, I will utilize transcriptomic signatures and high-throughput behavioral screening to identify and test candidate compounds that show promise in decreasing excessive alcohol consumption. This proposal provides a research and training plan for a transition from predoctoral FASD training to post-doctoral training in the biology of adult alcoholism, with the aim of developing an independent research program in the field of adult alcoholism.

NIH Research Projects · FY 2026 · 2019-06

SUMMARY: Diseases resulting in degeneration of the retinal pigment epithelium (RPE) are among the leading causes of blindness worldwide, and no therapies exist that can replace RPE or restore lost vision. Age-related macular degeneration (AMD) is one such disease, and is the third leading cause of blindness in the world. Transplantation of stem-cell derived RPE has emerged as a possibility for treating AMD and clinical trials are underway. An intriguing alternative approach to treat RPE disease is to develop therapies focused on stimulating endogenous RPE regeneration. Indeed, strategies targeting endogenous retinal regeneration are gaining traction as potential therapeutic approaches to treat a number of retinal degenerations. For this to become possible for the RPE, we must first gain a deeper understanding of the molecular and cell biological mechanisms underlying RPE regeneration. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but remaining RPE are unable to functionally replace lost cells when the lesions are large. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer. Moreover, RPE cells often overproliferate after injury, such as during proliferative vitreoretinopathy, where proliferative RPE cells invade the subretinal space and lead to blindness. A subpopulation of quiescent human RPE stem cells has been identified which can be induced to proliferate in vitro and differentiate into RPE or mesenchymal cell types. This discovery is exciting because it suggests that the human RPE contains a population of cells that could be induced to regenerate. Despite these studies, little is known about the process by which RPE cells respond to injury to elicit a regenerative, rather than pathological, response. There are also few models in which to study intrinsic RPE regeneration. This knowledge gap is a major barrier to developing effective strategies to restore RPE lost to disease or injury. To overcome this barrier, we developed a unique transgenic zebrafish model to study RPE injury and regeneration and leveraged this model to begin to identify the molecular and cellular mechanisms facilitating RPE regeneration. Our work to date supports our central hypothesis that injury-adjacent RPE cells proliferate in response to RPE damage to generate RPE daughter cells that then regenerate RPE lost to injury. Experiments in this proposal further test this hypothesis by i) determining how RPE heterogeneity influences RPE regeneration and identify the source of regenerated RPE; ii) determining the cell biological mechanisms underlying RPE regeneration; and iii) determining how the nrg1/ErbB pathway facilitates RPE regeneration. Understanding how injury-responsive RPE cells proliferate in vivo and the signals/pathways active during the injury response holds significant promise to identify strategies to stimulate or reactivate this ability in the human eye, which would be transformational for treating AMD and other diseases that affect the RPE.

NIH Research Projects · FY 2026 · 2019-05

PROJECT SUMMARY/ABSTRACT The high stability of local structure for RNA and DNA has profound and widespread impacts on life. The simplicity of base pairing provides a powerful strategy for enzymes and machines to recognize specific sequences, and structured RNAs use the high stability to fold incrementally. However, the ease and stability of base pairing also increases the odds and the consequences of misfolding, requiring RNA chaperones. More broadly, essentially all cellular processes that require structural rearrangements of RNA or DNA also require proteins to accelerate these rearrangements. This framework provides the broad theme of our research. Over the next several years, our focuses will be in three main areas. (1) Specificity of CRISPR-Cas enzymes. The overarching goal is to understand the molecular origins, strategies, and limits of specificity of these enzymes for their target sequences. We will explore the hypothesis that the affinity of the enzymes for their DNA or RNA targets is decreased by mismatches between the guide RNA and the target strand by an amount that can be understood from the intrinsic properties of the R-loop or RNA helix. With these affinity penalties as a starting point, we will explore the strategies that nature has used to generate specificity, and we will probe the origins of enhanced specificity in designed enzyme variants. (2) RNA chaperone activity of DEAD-box RNA helicase proteins. Over the past two decades, we have delineated how DEAD-box helicases can function as general RNA chaperones, using local RNA unwinding to promote folding and structural rearrangements of structured RNAs. Our goals for the next few years are to explore whether a helicase that functions as a general RNA chaperone can be converted to a specific chaperone by modular replacement of its intrinsically disordered C-tail. Preliminary results indicate that this substitution produces a functional chimeric protein, enabling the proposed structural and functional studies. (3) DNA and RNA compaction and folding. We will leverage a disulfide crosslinking approach that we developed in 2022 to build on our understanding of the electrostatics of nucleic acid helices and to probe RNA folding. We will extend our observations of trivalent cation-mediated attraction between DNA helices by testing polyamines and RNA helices, and we will extend the approach by measuring repulsion or attraction between nucleosomes. We will also adapt the approach to probe sharp bending of RNA junctions. In each research area, we strive to answer basic research questions that are likely to give important and generalizable insights. Our work also has implications for understanding and treating diseases, as defects in these proteins are linked to many diseases including cancer, and CRISPR-Cas enzymes have emerged as key tools to combat genetic diseases.

NIH Research Projects · FY 2025 · 2018-09

One in 10 Americans is living with diabetes, a chronic disease that occurs when the pancreas is no longer able to make insulin, or when the body cannot make good use of the insulin it produces. Diabetes impacts an individual’s quality of life, decreases their life expectancy, and increases their risk of heart disease, stroke, kidney failure, and dementia. These consequences of diabetes highlight the importance of identifying effective strategies for prevention of diabetes, and treatment and management of diabetes after diagnosis. In studies that aim to formally test such interventions, it is often the case that long term follow-up of patients is needed in order to observe the primary outcome. The identification and use of surrogate markers in such intervention studies have the potential to support more timely decision-making about the intervention’s effectiveness. While incredible progress has been made in the development of statistical methods to identify surrogate markers, existing methods for evaluating surrogate markers and appropriately using surrogate markers to test for a treatment effect in a future study still face key challenges. Importantly, there are no robust methods to identify when a surrogate may be useful for only certain subgroups of patients, where such subgroups are defined by multiple patient characteristics. Existing methods are limited to a single patient characteristic, but in practice, heterogeneity of a surrogate is more complex and is likely a function of multiple covariates. For example, the surrogacy of changes in hormone levels likely varies by not just sex but also age and body mass index. The impact of such complex heterogeneity is especially important to understand with respect to its potential to lead to violations of surrogacy assumptions that existing methods commonly require to ensure that future testing on the surrogate would accurately reflect testing on the primary outcome. When such testing is not in alignment, termed a surrogate paradox, incorrect conclusions about a treatment may be made. In this study, we aim to develop and apply robust statistical methods to address these challenges. Our methods, software, and results have the potential to inform and improve the design and analysis of future studies aimed at diabetes prevention by identifying when and how a surrogate marker can be used in future studies.

NIH Research Projects · FY 2025 · 2018-09

Project Summary Ribosomes are responsible for the rapid and accurate production of all proteins in cells in all forms of life on earth. The ability of these molecular machines to carry out faithful translation depends on their complex structure that allows dynamic interaction with ligands. The mature ribosome in eukaryotic cells is composed of two parts, the large 60S subunit that synthesizes all the proteins in a cell and the small 40S subunit that decodes mRNA. The production of a eukaryotic ribosome involves over 200 accessory factors which orchestrate the intricate processing and folding of the ribosomal RNA and assembly of the ribosomal proteins. Considering the complexity of ribosome structure and function and its critical role in decoding our genetic information, ensuring their correct assembly is a necessary but daunting task for cells. Lately, considerable interest has been focused on mechanisms of quality control in the ribosome biogenesis pathway. This proposal focuses on two distinct topics within ribosome assembly; (1) the mechanisms for assessing the structural and functional integrity of the newly assembled 60S subunit and (2) the transition from the early 90S pre-ribosomal precursor to the pre-40S precursor. With respect to assessment of structural and functional integrity, this proposal addresses three related questions: I. How are newly minted subunits assessed for function? II. What is the fate of defective subunits? III. What is the consequence of licensing defective ribosomes? In humans, defects in these processes lead to various diseases, including T-cell acute lymphoblastic leukemia and Shwachman-Diamond syndrome. Assembly of the small ribosomal subunit involves stepwise cotranscriptional assembly of the 90S particle, a large protein-RNA complex, scaffolded on U3-snoRNA. However, the presence of U3 is mutually incompatible with the final folded structure of small subunit RNA and must be removed once transcription of the RNA is complete and the 90S particle has fully assembled. The transition from the 90S to pre-40S is poorly understood. We propose that the displacement of U3 by the RNA helicase Dhr1 is a primary event that drives the transition of the 90S into the pre-40S particle. We will determine how the activity of Dhr1 is regulated to ensure the timely release of U3.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY/ABSTRACT Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide range of diseases. However, there is a fundamental gap in understanding how the global changes to the lipid environment affect local membrane protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein function, but it is difficult to determine the molecular mechanisms underlying protein-lipid interactions. The primary challenge is that conventional structural biology tools and binding assays are poorly suited to characterizing transient and heterogeneous protein-lipid interactions. To advance our understanding of biological process and lay a foundation for advancing disease treatment, our goal is to develop new approaches to determine how lipid bilayers regulate membrane proteins. Studying a diverse set of membrane protein targets ranging from bacterial complexes to viral ion channels to human transporters, we are focused on answering several key questions. First, which lipids bind a given membrane protein target? Lipids are often observed in membrane protein structures, but it can be challenging to determine the identity of the lipids present in the local lipidome surrounding membrane proteins. We will use novel lipidomic lipid exchange-mass spectrometry methods to study enrichment of specific lipid species in nanodisc lipoprotein particles containing the membrane protein target. Our goal is to identify unknown lipids that bind the membrane protein targets in complex mixtures of natural lipids. Second, how and were do lipids interact with the protein? We know that lipids can be critical for membrane protein function, but it is often unclear where and how specifically they bind. We will develop new native mass spectrometry methods to determine the sites and selectivity of lipid binding to membrane protein targets. Our goal is to uncover the molecular mechanisms driving lipid specificity at specific binding sites. Finally, why are lipids important for membrane protein function? We know that bunk cellular lipids are modulated in response to disease, age, and the environment, but it is unclear how these global lipid changes affect local membrane protein physiology. We will study the function of membrane protein targets in different lipid environments and with different mutants that affect lipid binding. For lipid sites that significantly affect function, we will perform structural analysis to connect lipid binding at specific sites with functional outcomes. Our overarching goal is to understand how global lipidomic changes affect local membrane protein structure and function. This will impact biomedical research by identifying lipids important for maintaining protein activity and aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimately, an improved understanding of protein-lipid interactions holds the potential for improved drug discovery and for new therapeutic strategies for modulating membrane protein activity by modulating cellular lipids.

NIH Research Projects · FY 2025 · 2018-08

This T32 renewal application aligns with the mission and values of The University of Texas at Austin’s (UT Austin) School of Social Work (SSW) and Dell Medical School (DMS) by providing a mechanism by which to enhance the pathways of future leaders in the sciences toward careers dedicated to promoting cardiovascular and respiratory health. The specific aims of the program are to: 1) recruit and provide rigorous scientific training to a cadre of developing scientists in order to increase the pool of highly-trained scientists prepared for a career in cardiovascular and lung research, and; 2) foster an environment at UT Austin for inter-professional development in cardiovascular and lung research. This renewal application proposes to continue to train 10 postdoctoral fellows for two years in the SSW and DMS. In addition, it proposes to expand the program to train 2 predoctoral fellows in the SSW. With involvement from 34 mentors in the Schools of Social Work, Medicine, and Nursing, the Population Research Center, and the College of Pharmacy, fellows will be trained in community-based participatory research methods and population health science. They will implement evidence-based interventions and/or produce scientific evidence with the ultimate goal of increasing health promotion in heart and lung diseases and their behavioral risk factors (e.g., dietary habits, physical activity, adherence, tobacco use/exposure, metabolic syndrome, obesity, sleep). Key innovations include recruitment and mentoring activities that include a team-based mentoring approach with a near-peer mentoring component, and cross-fertilization of social work and medical trainees. The DMS is uniquely designed to promote population health and the SSW has a strong community-based focus. Furthermore, the Schools have established a very successful and close partnership. Fellows will further their research training through formal mentorship, formal coursework, seminars, conferences, and other career development activities. Each fellow will have a primary mentor assisted by other mentors in a team-based approach, and a network of near-peers (postdoctoral fellows for predoctoral fellows and assistant professors for postdoctoral fellows) who will also contribute to mentoring. The Principal Investigators/Program Directors, Drs. Yessenia Castro, Catherine Cubbin, and Elizabeth Matsui will provide overall management of the program. An Advisory Committee, including external members, will assist them in the administration and evaluation of the program and its outcomes.

NIH Research Projects · FY 2026 · 2018-07

Project Summary/Abstract Post-traumatic stress disorder (PTSD) and major depressive disorder (MDD) are prevalent and disabling. Al- though trauma exposure is common and most trauma victims recover, the ~10% who develop chronic PTSD continue to have debilitating symptoms of re-experiencing, avoidance and hyperarousal as well as depression symptoms. It is critical to identify the underlying neurobiology of PTSD susceptibility and to further understand underlying differences between PTSD and MDD. Despite their clinical importance, there are limited human biol- ogy-focused postmortem brain studies of well-matched PTSD and MDD cases and controls to leverage the fact that these are well understood Psychiatric Disorders in terms of neural circuit regulation. This Competitive Re- newal utilizes a Collaborative R01 mechanism across 3-sites (University of Texas (1), Lieber Institute for Brain Development (2), and McLean Hospital (3)), to extend a postmortem, multi-omic study of brains (PTSD Brainom- ics Project), building on progress from data from the initial grant period with 300 additional new subjects in this proposal across the 3 diagnostic categories of 1) PTSD, 2) MDD, and 3) `neurotypical' controls. We will focus on targeted brain regions with known differential association with PTSD risk as a function of identified intermediate phenotypes, including amygdala, hippocampus, and dorsal raphe nucleus. We will conduct DNA genotyping (LIBD) and bulk-tissue DNA methylation and whole-genome bisulfite sequencing (on a subset), RNA-sequencing and protein expression across brain regions. We will also perform single nucleus (sn) Multiome-sequencing that combines snRNAseq and snATACseq to expand interpretation of the bulk multiomic data and spatial tran- scriptomics on select brain samples. Our new 900 samples from 300 new subjects will also support metaanalyses with prior samples for 1800 total brain samples from 600 total subjects. We will aim to investigate the roles of: 1) gene expression and alternative splicing in PTSD/MDD and trauma exposure in relation to upstream epigenetic regulation and downstream (phosphor)protein expression; 2) gene expression and chromatin accessibility in individual brain cells and spatial organization of molecular alterations in PTSD/MDD and trauma exposure; 3) polygenic risk and putative causal variants in PTSD/MDD and trauma exposed brains, and 4) to utilize systems biology, factor analysis and deep learning to understand traumatic stress related outcomes. We will use state- of-the-art statistical modeling, combined with rich psychological and biological phenotype measurements to de- termine disease-associated networks across brain regions and molecular pathways. This novel, integrated, and impactful linked R01 proposal will lead to identification of unknown trauma-associated genes and proteins, ncRNAs, and epigenetic marks in trauma related disorders and identify novel therapeutic targets. It will also expand understanding of cell-type-specific multiomic regulation. Our strategy has the potential to help redefine psychobiological subtypes of PTSD and reduce the burden of chronic PTSD.

NIH Research Projects · FY 2024 · 2017-09

There is a compelling need to decipher the role of the dysregulation of translation elongation in various chronic neurological conditions and many cancers. The long-term goal driving the proposed research is to help develop therapeutic strategies targeting eEF2K for treating progressive neurodegenerative diseases and malignancies. The overall objectives of this application are to (i) characterize the kinetic mechanism of eEF2K activation and regulation and (ii) elucidate the structural basis for the regulation of eEF2K by divalent cations, pH, ADP, and specific post-translational modifications. The central hypothesis is that calmodulin (CaM) binding activates eEF2K by profoundly altering its conformational dynamics, leading to a state capable of efficient phosphoryl transfer. Multiple regulatory inputs control the attainment of this state. The rationale is that understanding the mechanism of eEF2K regulation is necessary to provide a robust scientific framework for developing novel therapeutic approaches targeting neurodegenerative diseases and cancer. The central hypothesis will be tested through two specific aims: (1) to define the kinetic mechanism of activation of eEF2K by calmodulin and the kinetic basis for the modulation of its activity, (2) to determine the structural and dynamic basis of how specific inputs and post-translational modifications regulate the stability of active states of eEF2K. In the first aim, presteady state kinetics will define the precise allosteric mechanism of eEF2K activation and modulation of its activity. The second aim will determine the modulatory effects of specific post-translational modifications and other regulatory inputs in affecting the CaM sensitivity of the active state using a variety of structural approaches. The findings from both aims will be validated by characterizing eEF2K activation and activity in mammalian cells. In our opinion, the research proposed in this application is innovative because it focuses on understanding the relationships between the structural dynamics and the temporal control of eEF2K in mammalian cells using a unique combination of techniques integrated over multiple length scales from the atomic to the cellular. The proposed research is significant because it is expected to provide new insight into the cellular regulation of protein translation by eEF2K and provide critical advancement in understanding various eEF2K-driven pathologies.

NIH Research Projects · FY 2025 · 2017-08

SUMMARY Ribosome rescue pathways are conserved throughout bacteria, but the reason these pathways are important for physiology is not understood. The long-term goal of this project is to understand the function of ribosome rescue pathways and to target these pathways for new antibiotics. The overall objective of the proposed project is to identify interactions among components of the translation machinery that are specifically required for ribosome rescue and under what conditions different ribosome rescue systems are required. The central hypothesis of this work is that specific interactions within the ribosome and between the ribosome and other translation factors are uniquely required for ribosome rescue and that alternative rescue systems are critical under environmental conditions that cause RNA damage. The rationale for pursuing the proposed research is that it will determine why ribosome rescue is conserved in bacteria and will enable development of new antibiotics. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the molecular interactions required for trans-translation, 2) determine how ArfT rescues ribosomes in conjunction with either RF1 or RF2, and 3) determine why alternative ribosome rescue systems are required. Published work and preliminary data have identified small molecule inhibitors of trans-translation, and work in the first funded period of this grant identified their molecular targets. Biochemical and mutational analyses will be used in Aim 1 to determine why these targets are important for trans-translation and how the targets are disrupted by inhibitor binding. We will used structural and biochemical experiments in Aim 2 to determine the mechanism of a new alternative ribosome rescue pathway, ArfT, that can recruit either RF1 or RF2 to non-stop ribosomes. Our preliminary data identified conditions where the alternative ribosome rescue factor ArfB is required in Caulobacter crescentus, even when trans-translation is functional. We will determine the molecular basis for the ArfB requirement and determine of other alternative ribosome rescue factors are required under similar challenges in other bacteria. The use of small molecule inhibitors for chemical biology experiments to probe ribosome rescue is highly innovative, and the work proposed here is significant because it will delineate the physiological requirement for ribosome rescue pathways in bacteria and identify how these pathways can be inhibited.

NIH Research Projects · FY 2025 · 2017-08

Project Summary After carbon and hydrogen; oxygen, nitrogen, and fluorine are the three most common elements found in pharmaceuticals and drug candidates. Lead identification and optimization is typically the longest part of the drug discovery process, taking about five out of the average fifteen years, to bring a drug to market. This is partially due to the requirement to synthesize and test of a large number of potential drug candidates through structure activity relationship (SAR) studies. As such, the development of new organic methodologies for the rapid synthesis of organic molecules is of utmost importance to synthetic chemists. The research described herein focuses on the development of novel approaches for the synthesis of C–N, C–O, C–S, C–P, C–F, C–Cl, C–Br, and C–B bonds, in a selective and expedient manner. Moreover, it seeks to allow for divergent synthesis; where from a single common intermediate libraries of potential pharmaceuticals could be synthesized in a single synthetic transformation simply by varying the reagents. More specifically, the proposal focuses on the development of an array of carbon-carbon double bond difunctionalization of using a copper or palladium catalyst.

NIH Research Projects · FY 2026 · 2017-07

ABSTRACT This is a renewal application of an established program to investigate the regulation of the development and homeostasis of the spine. During the first funding period, our studies demonstrated that the gene Adgrg6, implicated in a common human spine disorder called adolescent idiopathic scoliosis, has an essential role in maintaining spine alignment in mice. We showed that the G-protein coupled rector Adgrg6 regulates gene expression and biomechanical properties of the intervertebral discs and dense connective tissue of the spine. Furthermore, we demonstrated that Adgrg6 stimulates cAMP signaling regulate factors essential for homeostasis of fibrocartilaginous tissue of the spine. Our findings suggest a new hypothesis that stimulation of cAMP signaling can decrease the onset and severity of scoliosis caused by the loss of Adgrg6 signaling. In addition, human genetics analysis of scoliosis identified a novel variant located in the transcriptional activation domain of the transcription factor SOX9. Significantly, targeted disruption of this domain of Sox9 in mice caused scoliosis and dysregulation of gene expression in fibrocartilaginous tissues of the spine. Here, we will test the hypothesis that Adgrg6 and Sox9 are functionally linked for regulation homeostasis and alignment of the spine. To add breadth to our program goals, we continued a forward genetic screen to isolate a collection of spine disorder mutant zebrafish. We recently identified two zebrafish mutants that fail to complement a novel thoracic scoliosis phenotype, suggesting a new pathway controlling spine morphogenesis. The characterization of this unique thoracic scoliosis phenotype will expand our knowledge into the cellular and molecular heterogeneity of spine disorders. Here, we will test the hypothesis that thoracic scoliosis in zebrafish is caused by a disruption of purinergic signaling leading to defects in notochord biogenesis. We will test these hypotheses via studies divided into three Specific Aims. Specific Aim 1 will deepen our mechanistic understanding of effectors of Adgrg6 signaling in the spine and test whether stimulation of cAMP can restore homeostasis to fibrocartilaginous tissues of the spine and halt the onset and progression of scoliosis. Specific Aim 2 will characterize the cellular and molecular causes of scoliosis in a novel Sox9 mutant mouse and use this model to test whether genetic interactions between Adgrg6 and Sox9 variants increase the susceptibility to scoliosis. Specific Aim 3 will characterize novel thoracic scoliosis mutant zebrafish and test a model that purinergic signaling is essential for notochord biogenesis and spine morphogenesis in zebrafish. Our results will provide new insights into the molecular genetics and biological processes necessary for the development and homeostasis of the spine. These studies may provide fundamental insights into the biological processes and pathways associated with human skeletal dysplasia and scoliosis.

NIH Research Projects · FY 2026 · 2017-05

Summary/Abstract While the human genome provides a parts list of >20,000 proteins, it is still largely unknown how these proteins assemble into ‘molecular machines’ to carry out their biological roles. This is important both for basic characterization of human genes and for understanding the mechanisms underlying most human genetic diseases, which often arise from defects in systems of proteins working together. We focus on the >9,000 human proteins shared across eukaryotes and dating to the last eukaryotic common ancestor. These ancient proteins carry out critical cellular processes, including DNA replication, repair, transcription, splicing, mitochondrial and ciliary processes, and trafficking, among others. They are disproportionately drivers of human disease, linked to a wide array of disorders, spanning cancers, birth defects, metabolic disorders, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and more. Nearly 1,300 of these deeply conserved human proteins are still mostly uncharacterized, despite almost certainly having important cell roles. A fundamental question is how all of these proteins work together to support cell function. However, a key limitation remains the lack of large- scale data directly interrogating these proteins’ expression, interactions, and activation states. Current approaches to quantify the proteome are only beginning to survey the proteins in mammalian cells to any significant depth, and consistently suffer from low sensitivity and throughput. These limitations have slowed medical applications, e.g. biomarker discovery, where techniques including mass spectrometry and antibody arrays often lack sufficient sensitivity and quantification accuracy to be effective. We propose research in three broad areas: First, we propose a major effort to biochemically define the main human protein complexes, providing a mechanistic basis for interpreting diverse human genetics and diseases. We will focus on evolutionarily conserved human proteins due to these proteins’ critical importance to cellular function, leveraging studies in other species using a comparative proteomics approach. Second, we are developing surrogate functional assays for deeply conserved human proteins by systematically humanizing yeast cells, replacing each essential yeast gene in turn by its human version. The resulting strains serve as new physical reagents for studying human genes in a simplified organismal context, opening up simple high-throughput assays of human gene function, the impact of human genetic variation on gene function, the screening and repurposing of drugs, and the rapid determination of mechanisms of drug resistance. Finally, we aim to advance new proteomics technologies, single-molecule protein sequencing and shotgun electron microscopy, both of which enable new types of highly sensitive characterization of protein expression and physical organization relevant to many aspects of human cell biology and disease. Success of these aims will give new insights into basic human cell biology and biochemistry, laying the foundation for future attempts to intervene, chemically or genetically, with those macromolecules most critical to the functioning of cells.

NIH Research Projects · FY 2025 · 2017-03

The long-term goal of our research is to understand how the properties of Cav channels shape their neural functions. The objective of this competing renewal application is to define the Cav1-dependent signaling pathways that shape the development and plasticity of the photoreceptor (PR) synapse. Among the major Cav1 subtypes expressed in the retina, Cav1.4 is uniquely critical for PR synaptogenesis. How Cav1.4 contributes to this process remains a mystery—a major challenge being that available animal models do not distinguish between the roles of Cav1.4 as a source of Ca2+ ions and as a scaffold for synaptogenic proteins. To overcome this hurdle, we generated a knock-in mouse strain expressing a non-conducting mutant form of Cav1.4. While the molecular organization of PR synapses is largely spared in these mice, the maturation of synaptic ribbons and invagination of postsynaptic neurites into PR terminals is disrupted. Our findings raise the intriguing possibility that the clinical variability associated with CSNB2 could arise from heterogeneous impacts of the mutations on the organization, development, and mature function of the PR synapse. Our central hypothesis is that Cav1.4 mediates Ca2+ signaling pathways that promote the maturation of synaptic ribbons and the postsynaptic architecture of PR synapses via mechanisms that are disrupted in CSNB2. We will test this hypothesis with the following Aims: (1) Elucidate the mechanism whereby Cav1.4 Ca2+ signals regulate the maturation and plasticity of synaptic ribbons (2) Define the role of Cav1 Ca2+ signals in enabling the postsynaptic wiring of PR synapses (3) Determine the impact of pathological variants of human Cav1.4 channels on PR synapse structure and function. The overall impact of our research will be knowledge of: (a) the multi-faceted roles of Cav channels at a synapse that is crucial for vision, and (b) how dysregulation specifically of Cav1.4 could lead to heterogeneous forms of vision impairment. More broadly, our research is expected to provide insights into mechanisms that enable the proper synaptic connectivity in the retina—a requirement for the successful restoration of vision through cell transplantation therapies.

NIH Research Projects · FY 2026 · 2017-02

PROJECT SUMMARY This is a competing renewal application for the Integrative Neuroscience Initiative on Alcoholism (INIA)- Neuroimmune consortium (Notice# RFA-AA-20-011, RFA-AA-20-013) to integrate multidisciplinary research projects based on the genomic, cellular, and behavioral neuroadaptations related to excessive alcohol consumption. This consortium has identified gene networks and pathways associated with excessive alcohol drinking in humans and animals and focuses on potential drug targets within neuroimmune and neuroinflammatory signaling pathways. In the next phase of this initiative, our collective proposals will address several documented NIAAA goals which include: 1) understanding the genomics, electrophysiology, and pharmacology of brain immune signaling systems in neurons and glial cells and their role in causes and treatments of alcohol dependence; 2) using new technologies such as single cell and spatial transcriptomics, proteomics, and multimodal functional and structural imaging to study these systems; 3) promoting reproducibility and translation of data through testing in multiple laboratories and in multiple assays; 4) guiding investigators in determining the translatability of their findings for preclinical and clinical studies by NIAAA- supported units outside the consortium. The overall hypothesis for INIA-N is that systematic analysis of neuroimmune mechanisms will inform strategies for treatment of excessive drinking associated with Alcohol Use Disorder. Ten Research Components and an Administrative Core comprise the consortium. INIA-N will be directed by the Administrative Core in cooperation with the Executive and Steering Committees and guided by a distinguished Scientific Advisory Board. The Administrative Core will provide leadership, oversight of scientific projects, and integration and translation of project data. INIA-N has six goals: 1) expand gene expression datasets with results from single nuclei sequencing and spatial transcriptomics to generate cell-type specific and anatomical transcriptome maps and integrate human cellular transcriptome data with human genome wide association studies; 2) define the contribution of specific non-neuronal cell types (astrocytes and microglia) to the molecular and behavioral effects of excessive alcohol consumption through a collaborative investigation of immune related cells of the brain; 3) examine alcohol-induced changes in perineuronal nets and in the abundance and post-translational modifications of extracellular matrix proteins as mechanisms for glial-neuronal cross talk that impact brain circuits regulating alcohol consumption; 4) pursue biochemical and electrophysiological studies of cytokine signaling to understand innate immune mechanisms by which excessive alcohol consumption changes brain function; 5) apply systems-level, connectomics approaches to identify mechanisms by which excessive alcohol consumption changes whole brain function, with emphasis on the role of our top neuroimmune genes; and 6) propose and prioritize drug targets and compounds for advancement to testing in animals and in humans by NIAAA supported entities outside the INIA-N consortium.