Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2018-08

Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. In particular, circadian clocks exert control over nearly every major metabolic pathway, allowing optimal utilization of typically cyclic availability of nutrients. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic metabolism. According to the current model, the core part of this clock mechanism is a negative feedback loop whereby the transcription factor heterodimer CLOCK/BMAL1 drives transcription of the “clock” proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 which interact with each other to repress the activity of CLOCK/BMAL1, and thus their own synthesis. This same transcriptional mechanism also drives rhythmic expression of many so called “clock-controlled genes” that ultimately result in the many output rhythms in tissues throughout the body. One of these clock- controlled genes is Nocturnin, a focus of my laboratory. We have shown that loss of this gene in mice causes resistance to diet-induced obesity, alters rhythms in cholesterol and triglyceride metabolism and increases resistance to inflammatory challenges such as LPS. Although the Nocturnin protein is a member of a family of RNases (the CCR4 deadenylases), we have recently demonstrated that Nocturnin’s substrate is not RNA, but rather NADP(H). Nocturnin is an NADP(H) phosphatase, converting NADP(H) to NAD(H). In this proposal we seek to use new mouse models that we have developed to understand the tissue-specific role of Nocturnin and to disentangle the roles of the two isoforms (one mitochondrial, the other primarily bound to intracellular membranes). We will also carry out metabolomic and metabolic flux experiments to determine the effects of circadian regulation of NADP(H) and NAD(H) levels. In addition, we will validate small molecule modulators of Nocturnin that we have identified in a high throughput screen and will use these molecules to perturb Nocturnin activity in cells and mice. Our work on the central mechanism of the core clock will also continue in the proposed funding period. We have solved crystal structures for the CLOCK/BMAL1 and CRY2/PER2 complexes and these data have allowed the identification of evolutionarily conserved functional domains throughout the proteins and revealed additional insights into the mechanisms by which these proteins operate and set the circadian period. Over the next five years, we will expand on this information to determine the atomic details of how this clock keeps time.

NIH Research Projects · FY 2025 · 2018-08

My laboratory has a long-term interest in understanding the basic mechanisms underlying actin cytoskeleton regulation and how these mechanisms drive fundamental processes such as cell migration, vesicle trafficking, immune cell activation, and neuron morphogenesis. We are particularly interested in solving the regulation mechanisms of the Wiskott-Aldrich Syndrome Protein (WASP) family proteins. These proteins play a central role in linking membrane signals to the Arp2/3-mediated actin polymerization throughout eukaryotic cells. Ge- netic mutation or misregulation of these proteins is heavily involved in human diseases, including various neu- rodevelopmental disorders, immune syndromes, pathogen infections, and cancer. Despite their importance, the regulation mechanisms of most WASP-family proteins are poorly understood, posing a large barrier to un- derstanding their roles in biology and human pathology and identifying new avenues of treating related dis- eases. Our recent studies have made a series of pivotal contributions to the understanding of the WASP-family protein WAVE and its role in disease. WAVE is incorporated in a large protein assembly named the WAVE Regulatory Complex (WRC), which is essential to polymerizing actin at the plasma membrane. We have deter- mined how WRC is activated by two distinct small GTPases, Rac1 and Arf, how disease-related mutations dis- rupt WRC activation, and how WRC interacts with various ligands, including the neuronal receptor HPO-30, the redox regulator p47phox, and the actin regulator Ena/VASP/UNC-34. In the next five years, we will continue to move the field vertically by addressing a series of new, important questions about WAVE regulation. In parallel, we will start exploring the mechanisms of two other WASP-family proteins, WHAMM and JMY, for which very little is known mechanistically despite their essential roles in regulating actin assembly at endomembranes. We will combine protein engineering, quantitative biochemistry and mass spectrometry, single molecule fluores- cence microscopy, and single particle cryogenic electron microscopy (cryo-EM) to determine how WRC is acti- vated by Arf binding, how distinct Arf and Rac1 binding sites establish cooperativity, and how WRC interacts with several novel ligands important to neuron morphogenesis, axon guidance, and parasite infection. In addi- tion to dissecting mechanisms of individual ligands, we will establish membrane-based single-molecule fluores- cence experiments to determine the mechanisms by which multiple ligands, including inositol phospholipids, GTPases, and membrane proteins, cooperatively activate the WRC in a context closely resembling cell mem- branes. Furthermore, we will determine how WHAMM and JMY are regulated by binding to various ligands. Our work will provide a comprehensive mechanistic framework for understanding WASP family protein regula- tion. This knowledge will be broadly useful to different fields studying actin-related processes. In collaboration with various biologists and clinicians, our work will reveal how mutations in WASP-family proteins derived from patients disrupt function and provide new ideas and targets for the development of novel intervening agents.

NIH Research Projects · FY 2025 · 2018-08

Project Summary CRISPR/Cas-based gene editing has ushered in a hopeful era that dreams of new therapies for currently untreatable genetic diseases. Because mutated proteins are produced in specific cells, there is a critical need to develop organ- and cell-specific delivery strategies to realize the full potential of genomic medicines. We recently overcame this challenge through development of the first class of non-viral nanoparticles for tissue-specific genome editing. Selective ORgan Targeting (SORT) lipid nanoparticles (LNPs) enable targeted intravenous delivery of nucleic acids and proteins to the lungs, liver, and spleen, plus local delivery to the muscle, brain, and skin. Tropism is driven by inclusion of SORT molecules, which create tissue-selective 5-component SORT LNPs that are compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes. In this grant proposal, we Aim to (1) determine the mechanism of SORT, (2) improve the efficacy and tolerability of liver-, lung-, and spleen-targeting SORT LNPs, and (3) determine the cell-specific gene editing capabilities of SORT LNPs with the potential for expanded tropism. Results will determine the fundamental mechanisms and structure-activity relationships (SAR) for non-viral nanoparticle liver, lung, and spleen tropism. This will ultimately allow targeted and safer CRISPR/Cas gene editing in vivo. We will determine these factors by adapting a unique class of LNPs, called SORT LNPs, that we developed. We will employ human cells and genetically engineered mouse models that allow quantification of precise, cell specific gene editing events. Completion of the proposed studies will (1) Elucidate the fundamental mechanisms how and why SORT LNPs target extrahepatic tissues, (2) Determine how SORT molecules control efficacy and tolerability for improved gene editing outcomes, and (3) Determine and control cell-type gene editing specificity to expand targeted gene editing. Cumulatively, this will open new avenues for CRISPR/Cas-based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle carriers.

NIH Research Projects · FY 2025 · 2018-08

Alcohol-associated liver disease (ALD) is a leading cause of liver-related mortality/morbidity, and there is no FDA-approved therapy for any stage of ALD. Advanced ALD conditions, including severe alcohol-associated hepatitis (sAH) have especially poor outcomes. Indeed, the 90-day mortality for sAH is ~30%. Return to drinking impacts quality of life, morbidity and mortality in these patients. There are limited drug therapies or well-studied behavior therapies in this patient population. An optimal approach would be the integration of AUD and ASLD care givers and therapies, but there are no guidelines for this approach. Our proposed AUD/ALD team approach seeks to overcome the perceived stigma of alcohol misuse which can adversely affect treatment seeking, quality of care and patient outcomes. The AlcHepNet RCT was stopped at the interim analysis because of the unexpected 90% 90-day survival in sAH patients treated with prednisone using the Lille stopping rule. These dramatic results need to be confirmed, and novel therapies such as IL-22 need to be studied in sAH. Acamprosate appears to be the safest FDA-approved therapy for AUD in patients with ALD, but safety and efficacy in severe ALD need to be evaluated. Motivational interviewing/enhancement therapy is well-suited behavioral therapy for patients with ALD. Based on preliminary data and knowledge gaps, our overall hypothesis is that integrated management of ALD and AUD will improve clinical outcomes in patients with sAH and decompensated ALD. We will utilize the following AIMS: Aim 1. Perform a randomized controlled trial of treatment for steroid-eligible patients with severe AH. A SMART trial design will be used to compare daily prednisone for 28 days (with the 7-day Lille score-based stopping rule) vs IL-22 fusion protein (F-652) followed by randomization of each of these groups to receive motivational Interviewing/enhancement therapy combined with acamprosate vs usual care including referral to 12-step programs before discharge from the hospital. The primary endpoint of the trial will be a composite measure of mortality, liver, and alcohol use related outcomes at 6 months. AIM 2. Build a platform for biosamples, data repositories, and patient registries to support site- specific and network-wide ancillary studies. In summary, these proposed studies will leverage the existing resources of the AlcHepNet to evaluate the clinical impact of integrated ALD/AUD treatment in a cohort of patients for whom there are limited treatment options.

NIH Research Projects · FY 2025 · 2018-07

Project Abstract/Summary The rising prevalence of obesity and type 2 diabetes threatens to limit human healthspan by increasing the risks for cancer and cardiometabolic disease and to impose overwhelming economic burdens. New therapeutic strategies are urgently needed. Since the discovery of functional brown and beige adipocytes in adult humans, much attention has focused on exploiting the ability of these thermogenic adipocytes to dissipate excess energy as heat through uncoupled mitochondrial respiration. Advanced imaging in humans has revealed a favorable correlation between brown fat mass and cardiometabolic risk factors. A major gap in the field is a safe and effective pharmacological strategy to activate brown and/or beige adipocytes to promote negative energy balance, reverse obesity, and mitigate obesity-related metabolic disorders, such type 2 diabetes, cardiovascular disease, and nonalcoholic fatty liver disease. The overall goal of this application is to provide proof-of-concept for one such strategy that has been suggested by the lab’s long- standing research program on Perilipin 5 (PLIN5), a member of the Perilipin family of lipid droplet proteins that is expressed in oxidative tissues, including brown adipose tissue (BAT). A growing body of literature from our lab and others has implicated PLIN5 not only in the regulation of lipolysis at the lipid droplet surface, but also in the regulation of gene expression via interactions in the nucleus with SIRT1 and PGC1a and in the tethering of lipid droplets to mitochondria. Our published work in mice has shown that PLIN5 is required for the metabolic, transcriptional, and mitochondrial adaptations of BAT to cold stress. We have also shown that PLIN5 gain-of-function in BAT of mice can prevent glucose intolerance and fatty liver from high-fat diet and promote healthy remodeling of white adipose tissue (WAT) with prevention of adipocyte hypertrophy. In this renewal application we propose to test the hypothesis that promoting PLIN5 expression in BAT in conjunction with b3 adrenergic receptor agonist treatment of diet-induced obese mice will reverse adipocyte hypertrophy in WAT, reverse obesity, and reverse glucose intolerance. Aim 1 will elucidate the metabolic and signaling pathways responsible for the effects of PLIN5 on mitochondrial form and function in BAT and on systemic lipid and glucose metabolism by means of genetic mouse models, in vivo structure- function studies, and mechanistic mitochondrial experiments. Aim 2 will interrogate the signaling pathways and physiological responses associated with treatment of diet-induced obesity with a 2-hit intervention built on augmentation of PLIN5 in BAT in synergistic combination with b3 adrenergic receptor agonist treatment. Successful completion of these Aims may establish the conceptual foundation for a new therapeutic paradigm for treatment of obesity that is efficacious but at lower, non-toxic doses of existing medications.

NIH Research Projects · FY 2026 · 2018-04

PROJECT SUMMARY Antibiotic resistance is one of the most serious public health challenges of our time. In this proposal, we focus on two common antibiotic resistance mechanisms that are: (i) alteration of drug target enzymes and (ii) modification of antibiotic molecules. Despite recent advances in biological sciences, clinically accessible antibiotics can still target only a handful of enzymes, such as DNA gyrases and dihydrofolate reductase (DHFR). Hence, it is important to better understand molecular evolution of these enzymes and accordingly develop effective drugs that target these enzymes without exacerbating the resistance problem. Similarly, modification of -lactam antibiotics through hydrolysis by -lactamases is currently the most concerning antibiotic resistance mechanism because -lactams account for nearly seventy percent of antibiotics currently used in clinics. To address this important health problem, we propose an innovative research plan to study evolution of the DHFR enzyme, and develop mutant-specific competitive and allosteric DHFR inhibitors to select against resistance- conferring DHFR mutations. Finally, to address the -lactam resistance problem, we will engineer a novel class of molecules that we named “-lactamase traps” (or BLTs) to select against -lactamase producing bacteria. Our first aim is to map epistatic interactions that shape DHFR evolution under antibiotic selection. DHFR is a ubiquitous enzyme with a central role in metabolism. We have previously shown that E. coli DHFR accumulates 3 to 5 resistance conferring mutations following a quasi-deterministic order, as a result of strong epistatic interactions between DHFR mutations. We will quantitatively map all epistatic interactions in the DHFR fitness landscape by using state-of-the-art genetic tools and the Hierarchical Model, a mathematical toolbox we developed to efficiently explore the fitness space and identify epistatic interactions between mutations. Our second goal is to engineer competitive and allosteric mutant-specific DHFR inhibitors. We have previously developed an L28R-specific competitive trimethoprim derivative (4’-dTMP) that impeded evolution of resistance by selecting against the L28R mutation. Following a similar procedure, we will engineer new competitive DHFR inhibitors that will target resistance-conferring mutations on the D27, W30, I94, and F153 residues. Similarly, we will identify allosteric inhibitors that can target a druggable DHFR cryptic site we discovered. This site has limited drug accessibility for the wild-type DHFR but always remains open for the D27E, I94L, and F153S variants of DHFR. We already showed that proglumetacin, a commonly used NSAID drug, allosterically slows down DHFR activity. Our third goal is to engineer a novel class of molecules that select against -lactamase genes. We will develop a novel class of molecules that we call BLTs to eliminate -lactamase producing Gram-negative bacteria. BLT molecules are inactive in their native form but once activated by -lactamases, turn into potent antibiotics. We have already designed and synthesized a cephalosporin-based BLT molecule that is activated by the CTX-M cephalosporinase. We will generate a library of cephalosporin- and carbapenem-based BLT molecules.

NIH Research Projects · FY 2025 · 2018-04

PROJECT SUMMARY How skilled behaviors like speech and language are actively maintained throughout life is not well understood and still poorly studied. Our research program uses songbird vocal learning and vocal production to understand how forebrain circuits and reinforcement mechanisms are used to acquire and then maintain learned vocalizations. Our research has helped demonstrate that midbrain dopaminergic circuits bidirectionally guide learned changes in song in a manner consistent with them functioning as reward prediction error signals envisaged by reinforcement models. Building from this, we turn our attention to understand how the basal ganglia and dopaminergic circuits support the lifelong maintenance of behavior. We hypothesize that predictive dopaminergic signals safeguard the lifelong maintenance of natural behaviors. Our initial studies provide a glimpse at central mechanisms sufficient to initiate the long term decrystallization of a previously learned and internally reinforced natural behavior. Using a variety of cutting-edge approaches that we have optimized for circuit interrogation in songbirds, we aim to dissect the cellular and synaptic mechanisms associated with song decrystallization, and the role of circuit nodes downstream of dopaminergic pathways in song maintenance and song decrystallization. In the first aim we will test the role of predictive dopaminergic signals in the long-term maintenance of adult zebra finch song using optogenetic manipulations and functional imaging of dopamine activity in adult animals. In the second aim we will examine the cellular and synaptic mechanisms of song decrystallization. In the third aim we will test the role of pallidal-thalamic circuits downstream of dopaminergic striatal pathways in the implementation and rescue of song decrystallization. Together, these studies can provide fundamental and mechanistic insights into how the brain continuously monitors and updates behavior to maintain expert performance and reveal what happens when this process goes awry.

NIH Research Projects · FY 2026 · 2018-04

PROJECT SUMMARY Glutamine is a conditionally essential amino acid that has myriad uses in the cell. Aside from direct incorporation into protein, glutamine can be metabolized to generate nucleotides, other amino acids, the Krebs cycle intermediate α-ketoglutarate (αKG) which is important for energy production and anabolic reactions, and glutathione (GSH) to protect against oxidative stress. We recently determined that αKG and GSH are particularly important glutamine derived metabolites that are required at distinct stages of osteoblast differentiation. For example, our data indicates that αKG is required early in skeletal stem and progenitor cells to regulate proliferation and osteoblast specification. GSH is required later in specified pre-osteoblasts to govern osteoblast differentiation by neutralizing reactive oxygen species (ROS). Mechanistically, how αKG and ROS regulate proliferation, specification and differentiation remains enigmatic. Moreover, the enzymes that generate αKG from glutamine in osteoblasts have not been described. In this proposal, we will 1) establish the necessity of glutamate oxaloacetate transaminase 2 (GOT2) dependent αKG synthesis to regulate osteoblast differentiation and bone formation, 2) define the contribution of αKG to osteoblast energetics and intermediate metabolism, 3) elucidate the molecular regulation of osteoblast differentiation by ROS, and 4) evaluate the efficacy of antioxidants to rescue bone formation in a mouse model of human cleidocranial dysplasia. Our findings will have broad implications in bone development, maintenance of bone mass, skeletal repair and regeneration.

NIH Research Projects · FY 2025 · 2018-04

Project Summary/Abstract This application seeks to establish a T35 NEI Summer Research Program to enhance medical student research training in the prevention and treatment of eye diseases at UT Southwestern Medical Center. The proposed program will provide short-term training opportunities for medical students in basic and clinical biomedical research focused in areas supported by the NEI. Based on our extensive experience gained from other NIH Research Training grants, we will develop a short-term training program for those medical students enrolled at UT Southwestern as well as outstanding applicants from medical schools across the country. Building upon our collective experience in the identification and pairing of students and mentors, we anticipate that these experiences will continue to enrich the research background of participating medical students with an interest in eye diseases. Besides the mentor-based teaching within the laboratories and clinics, trainees will also receive a comprehensive course in Research Methodology with NEI-specific journal clubs. The program described in this application will employ the outstanding faculty and resources already in place at UT Southwestern that are focused in areas of NEI interest. This program will serve as a focal point to further enrich medical student research activities at UT Southwestern and will enhance existing elements of NEI-funded research on campus.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY The goal of our project is to investigate the molecular mechanisms of induction and decline of meibogenesis in Meibomian glands (MG) embedded in tarsal plates of humans and mice. Meibogenesis is defined as an intricate array of catabolic and anabolic reactions, and corresponding regulatory and signaling mechanisms, that lead to formation of a holocrine secretion called meibum. Meibum is a unique lipid secretion that is comprised primarily of extremely long chain and branched wax esters, cholesteryl esters, and a range of other, more complex, compounds. Meibum is vital to the ocular health as it forms a protective layer that isolates the surface of the eye from the environment, and improves vision by changing the refractive properties of the cornea. Lipid composition of meibum is very conservative in normal conditions, implying that lipid homeostasis of MG is typically under tight control of yet to be identified regulatory mechanisms. However, a MG pathology called MG dysfunction (MGD) results in a decline in meibum production, or adverse changes in its composition, or both, negatively affecting the ocular surface physiology, vision, and quality of life in general. MGD is a major contributing factor to a widespread condition called Dry Eye syndrome (DES). MGD and DES affect up to 40% of the general population worldwide, disproportionately affecting elderly. Earlier, we demonstrated that mice are credible models of human MG for studying meibogenesis. Using various lines of mutant mice, we have established major genes and enzymes that are involved in meibogenesis. However, the mechanisms of its initiation and regulation remain unknown. Previous attempts to induce meibogenesis in cell cultures (such as immortalized human MG epithelial cells) failed, as no meibomian lipids have been produced in any tested conditions. Thus, our aim is to elucidate the mechanism of meibogenesis induction and decline in vivo by conducting transcriptomic, lipidomic, immunohistochemical, and physiological characterization of developing and aging MG, using mice that undergo prenatal and postnatal development and aging as primary animal model, and human subjects of different ages. These experiments should allow us to determine a timeline of changes in developing, maturing, and aging MG, and correlate MG transcriptome in general, and key genes of meibogenesis specifically, with the expression levels of specific enzymes and their corresponding lipid products. Special consideration will be given to genes that simultaneously: 1) are highly expressed in MG, 2) encode signaling factors that are already known to control tissue growth, cell differentiation and lipid homeostasis in MG and/or other tissues, and 3) whose expression levels undergo significant changes in developing and aging MG. These results will provide critically important information for future in-depth studies of MG physiology in the norm and pathology.

NIH Research Projects · FY 2025 · 2017-09

The Resource for Molecular Imaging Agents in Precision Medicine is a transdisciplinary consortium of facilities and expertise centered at Johns Hopkins University. Participants are the Russell H. Morgan Department of Radiology and Radiological Science, the F. M. Kirby Research Center at the Kennedy Krieger Institute, the Sidney Kimmel Comprehensive Cancer Center, and the Department of Biomedical Engineering, with key industrial collaborators in biomedical imaging and pharmaceuticals to ensure widespread dissemination. Our work will synthesize, develop, and deploy precision imaging tools and theranostic agents for early detection, interception, and cure. The long-term objective of the NCBIB is to promote the translation and dissemination of new molecular imaging and theranostic agents and their attendant best practices along the spectrum of cancer, inflammation and immunity. Our proposed work capitalizes on advances made in the past five years in artificial intelligence (AI), new and more sensitive translational imaging devices, nanotechnology, gene manipulation, and new techniques to produce specific molecular affinity agents and will create a scientific ecosystem that can transform the healthcare landscape. Collaborative academic centers such as ours, while steeped in the culture of fundamental discovery, are beginning to pivot toward the development of new work products – working backwards from unmet needs – in the context of increasing academic-industrial partnerships and entrepreneurship. Working closely with our partners, we will leverage these advances in scientific and academic cultural thinking, and provide new materials to our collaborating and service partners, some of whom have worldwide reach, to address pressing and unsolved medical challenges. During the first funding period we consolidated our ability to work together seamlessly as a NCBIB; in this renewal, we have added collaboration with a new local NCBIB that complements our work by providing biological reagents relevant to immunoengineering, offering AI capability, founding one company dedicated to advancing use of AI in molecular imaging and another company for commercialization of imaging and theranostic agents, received FDA approval for an imaging agent (analogs of which are already in use in the NCBIB), and providing precursors, other reagents and IND cross-references to multiple institutions. Our goals for the renewal period are, across four TR&Ds, to develop: new reagents to detect and promote an immune reactive tumor microenvironment (TR&D1); an integrated nanoplatform to manage a variety of cancers (TR&D2); translational imaging agents, theranostics, and software for managing inflammation and/or cancer in the periphery or central nervous system (TR&D3); and a method to use extracellular vesicles as a nanotheranostic platform in neuroinflammation (TR&D4). Together with the Collaborating and Service Projects (CPs and SPs) we will generate next-generation precision platforms, tools, and techniques for tackling problems at the forefront of biomedical research with a focus on those that will lead to near-term translation, as we have done previously.

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY/ABSTRACT Metabolic reprogramming is a hallmark of malignancy and potential source of therapeutic targets. Recent work indicates that metabolic liabilities change as cancer progresses, meaning that the pathways most relevant to advanced cancers may not be apparent in locally-invasive, treatment-naïve tumors at the site of origin. Recognizing the dearth of direct information about human cancer metabolism, we developed an approach to probe the metabolic network of intact human tumors by infusing patients with stable isotope-labeled nutrients (e.g. 13C-glucose) during tumor resection or biopsy. By measuring isotope labeling in metabolites extracted from tumor samples and following the outcomes of patients who underwent this procedure, we identified metabolic properties associated with poor survival. Of hundreds of metabolic features, 13C labeling in tricarboxylic acid (TCA) cycle metabolites was the most predictive of cancer progression and early death. In non-small cell lung cancer (NSCLC), patients whose tumors have high labeling of these metabolites succumb much earlier than patients with low labeling, and blocking this pathway in mouse models of NSCLC suppresses metastasis. In clear cell renal cell carcinoma (ccRCC), TCA cycle labeling is low when tumors are localized to the kidney but much higher in metastatic tumors, and activating the TCA cycle promotes metastasis in mice. Therefore, in both kinds of cancer, data from patients lead us to conclude that oxidative mitochondrial metabolism, particularly the TCA cycle, electron transport chain (ETC) and oxidative phosphorylation (OxPhos), promote cancer progression. The success of these experiments prompts us to further study the metabolic basis of human cancer progression in the hopes of developing new insights and therapies. We propose three general directions. First, using a combination of approaches in humans and mice, we will thoroughly examine how mitochondrial metabolism stimulates metastasis to identify discrete metabolic dependencies that could be safely targeted in patients. Second, we will develop approaches to discover new metabolic liabilities in human tumors. Strategies include a pipeline to probe viable tumor explants with a series of isotope-labeled nutrients under physiological conditions to choose the most informative tracers for isotope infusions in patients; and dynamic imaging methods to observe and quantify informative aspects of metabolic flux in tumors in real time. Third, we will use the orthogonal approach of studying human inborn errors of metabolism (IEMs) to discover why some metabolic anomalies prime cells to become malignant. This approach capitalizes on a clinical cohort of over 1,000 subjects, including patients with IEMs associated with highly penetrant cancers, and will provide unique insights into cancer initiation and progression. Altogether these efforts will build on our long-standing productivity in human cancer metabolism by uncovering new mechanisms governing the metabolic basis of cancer progression and producing new methodologies to understand and treat lethal malignancies.

NIH Research Projects · FY 2026 · 2017-08

Mammalian development results in the specification of over 200 different cell types from a single genome, with subsequent maintenance of cell identity in adult organisms. The genomes of eukaryotic cells are packaged into a dynamic chromatin structure, which allows cells to control the accessibility of all DNA encoded information. The selective incorporation of specialized histone proteins, or variants, into this dynamic genomic structure is an important feature of epigenetic regulation. A main focus of my lab is one such protein, the histone variant H3.3. The identification of mutations in H3.3 and associated proteins in human cancers and developmental disorders has heightened the pressing need to understand the role of this histone variant in normal development and adult homeostasis. Although H3.3 is critical to cellular function in multiple contexts, how H3.3 contributes uniquely to chromatin function is a long-standing, unanswered question in the field. While long associated with gene activation, recent studies establish that H3.3 also deposited at repetitive, heterochromatic regions of the genome, with deposition at each region facilitated by independent chaperone complexes. We still do not know how H3.3 is partitioned between its two chaperone complexes, or how this equilibrium influences cellular function. Once deposited, our studies and others have demonstrated that H3.3 influences the chromatin modification landscape at both euchromatin and heterochromatin. Our data suggest that H3.3 can perform this function directly at euchromatin via phosphorylation of a unique serine that influences the activity of a histone acetyltransferase or indirectly at heterochromatin through chaperone-mediated recruitment of a co-repressor complex. Despite these intriguing observations, we do not yet fully understand the detailed mechanisms by which H3.3 deposition influences chromatin states. Finally, we do not understand how H3.3 performs its myriad functions in the context of complex, multicellular organisms. The goals of this proposal are to: (1) understand how H3.3 chaperone complex equilibrium is established, and determine the effects of disequilibrium on cell function, (2) determine the molecular mechanisms by which H3.3 influences local chromatin landscapes, and how these events influence downstream genome usage, and (3) make use of our novel mouse models to understand the role of H3.3 in adult organisms, including adult stem cells. Our proposed research is significant because it will serve as a platform to understand epigenetic regulation of cell identity in both normal development and adult homeostasis, and by extension, developmental misregulation and disease states. Project Summary/Abstract

NIH Research Projects · FY 2026 · 2017-07

Hypoxic-ischemic encephalopathy (HIE) is the leading cause of newborn death and disability worldwide and presents clinically as neonatal encephalopathy that is difficult to classify within a short window after birth to inform neuroprotective interventions. Therapeutic hypothermia (TH) has dramatically improved outcomes in moderate to severe HIE when initiated within six hours of life (HOL), yet trials have not included mild HIE. The difficulties to discern the clinical severity shortly after birth and to analyze the dynamic circulation in sick newborns represent important challenges. As new trials are now considered to target unstudied mild HIE, neurophysiological and neuroimaging biomarkers are critically needed 1) to guide real-time patient selection within a short therapeutic window and 2) to provide insight into timing of the metabolic energy failure and effect on structural and functional brain outcomes. Our team pioneered a “wavelet neurovascular bundle” analytical system to measure neurovascular coupling (NVC) under dynamic conditions and in real time, and to measure regional oxygen consumption one step further into the core of brain energy homeostasis. The parent grant initially focused on infants with moderate to severe HIE during the 72 hours of treatment and resulted in twenty six new publications. The overarching goal of this renewal is to harness our novel physiological biomarkers to focus on untreated mild HIE to identify those who need treatment. Our objective is to test dynamic biomarkers that can predict structural and functional outcomes chosen with input from stakeholders, and inform physiological and metabolic disturbances specific to mild HIE. We plan to enroll a new cohort of 100 neonates with mild HIE (35 per year over 3 years) and to follow up for two years infants for developmental outcomes. Our infrastructure ensures the success of this proposal and includes a multidisciplinary team of experts in hemodynamics and electrophysiology signals (Chalak, Zhang), bioengineering (Liu), and neuroimaging (Liu P, Wisnowski). The high-volume, supportive research environment at Parkland Hospital, including a 3T Siemens Skyra located inside the NICU, permits imaging of sick newborns as early as the first day of life. New knowledge will specifically provide: 1) Development and optimization of neuroimaging and neurophysiological assessments of mild HIE; and 2) improved HIE stratification via multi-modal assessments. Impact. The ability to monitor global neurovascular functions in real time could lead to a paradigm shift by providing the field with sensitive physiological biomarkers to detect the evolution of deficits in mild HIE and allow targeted interventions in this unstudied population. Engaging community stakeholders ensures that the NIH mission remains aligned with the priorities of affected families.

NIH Research Projects · FY 2025 · 2017-07

Modified Project Summary/Abstract Section SUMMARY: Childhood obesity is one of the most common pediatric chronic diseases in the US. We have shown (HL136643) that over one year, children with obesity (CWO) can add four times as much fat weight as children without obesity (CWOO). However, it is unknown if this rate of increase in fat weight continues into adolescence, and whether respiratory function, exercise tolerance, or dyspnea on exertion (DOE) are progressively worsened by increasing obesity. Furthermore, there could be a sex difference in the effects of obesity, given the different growth characteristics for boys and girls. The overall objective of this proposal is to investigate the changes in body composition, lung function, exercise tolerance, and DOE after 6 years of aging in CWO and CWOO. In CWO, excess fat exerts an unfavorable burden on the respiratory system, particularly during exercise, potentially reducing exercise tolerance and leading to DOE. We have found that most of the respiratory effects in CWO are the result of low lung volume breathing, which exposes children to breathing limitations like expiratory flow limitation, gas exchange impairment, and greater perception of dyspnea. Our study approach will be to re-examine body composition, respiratory function, exercise tolerance, and DOE in CWO and CWOO who were originally studied as 8–12-year-olds (i.e., originally Tanner score ≤ 3; 90 participants; 26 CWOO & 64 CWO). Specific Aims: We will test the following hypotheses: Aim 1) CWO originally studied at 8-12 years old will demonstrate a greater increase in fat weight and lower respiratory function (i.e., altered pulmonary function & breathing mechanics at rest) than in CWOO originally studied at 8-12 years old; Aim 2) CWO originally studied at 8-12 years old will demonstrate lower exercise tolerance measured during graded cycle ergometry (peak V•O2 in ml/min/kg) than in CWOO originally studied at 8-12 years old, but not lower cardiorespiratory fitness (peak V•O2 in % of predicted based on ideal body wt.); and Aim 3) CWO originally studied at 8-12 years old will demonstrate greater DOE as evidenced by increased ratings of perceived breathlessness during constant load exercise cycling than in CWOO originally studied at 8-12 years old. These novel longitudinal results will have broad mechanistic relevance to our understanding of the effects of worsening obesity as children age.

NIH Research Projects · FY 2026 · 2017-07

PROJECT SUMMARY Antipsychotic drug (APD)-induced metabolic syndrome is a pressing clinical problem affecting millions of patients. However, the difficulty in modeling their metabolic effects in laboratory animals has significantly hindered relevant mechanistic studies. To this end, we have developed new mouse models that recapitulate human metabolic syndrome caused by two commonly prescribed APDs (olanzapine and risperidone). Metabolic analyses revealed that drug-induced hyperphagia is the driving force behind weight gain in both models. Using bulk RNA sequencing, we investigated how APDs altered gene expression in the hypothalamus—a brain region that is critical for appetite control. Our analyses revealed that the melanocortin 4 receptor (Mc4r) was among those that were directly regulated by APD treatment. Furthermore, we found that the obesogenic effect of olanzapine and risperidone depends on Mc4r in Sim1 neurons. Moreover, we found that APDs reduced hypothalamic Mc4r mRNAs before the weight gain. Remarkably, whole-cell electrophysiology experiments demonstrated for the first time that olanzapine and risperidone acutely inhibited Mc4r-expressing neurons in the paraventricular nucleus of the hypothalamus. Furthermore, this inhibition was mediated by a postsynaptic potassium conductance. Collectively, these findings provided the first experimental evidence linking deficits in hypothalamic MC4R signaling to APD-induced metabolic syndrome. In the current project, we propose a multi-discipline approach to investigate the mechanisms underlying 1) how olanzapine and risperidone interact with MC4Rs and perturb their functions; 2) how they inhibit the activity of Mc4r neurons; 3) how both drugs alter the transcriptional and chromatin landscapes in hypothalamic neurons at the single-cell level. These studies have important clinical implications based on the suggestions that MC4R can be a novel therapeutic target for APD-induced weight gain, and that they may guide the development of next-generation antipsychotic medications with fewer metabolic side effects.

NIH Research Projects · FY 2025 · 2017-07

Heart failure (HF) disproportionately affects older adults, who carry a high burden of cardiovascular risk factors and experience accelerated decline in cardiac function. Subsets of older adults do not experience progressive cardiac dysfunction, but the factors related to late-life cardiac resilience are not well defined. This is a critical barrier – and missed opportunity – to identify novel interventions and treatment targets to prevent HF. The objective in this application is to define the lifestyle factors, social drivers, and molecular pathways underlying cardiac resilience in very late life. The central hypothesis is that in addition to optimal risk factor control, salutary health behaviors (exercise, diet) and favorable structural factors (less social adversity) protect from age-related cellular senescence (assessable via plasma proteomics) and promote trajectories of preserved cardiac function in late life. Leveraging rich longitudinal phenotypic data – including echocardiography – of participants in the community-based Atherosclerosis Risk in Communities (ARIC) study, sequential echocardiography will be performed in ~2,175 participants attending the 12th study visit (age ~86±4), in addition to functional assessments and measurement of plasma proteomics. The resulting three serial echocardiograms over 12 years will be used to identify trajectories of change in cardiac function and to address the following aims: (1) Define late-life behaviors and factors associated with cardiac resilience; (2) Identify proteins and protein networks underlying cardiac resilience with particular attention to circulating markers of cellular senescence; and (3) Determine the association of cardiac resilience with preservation of physical and neurocognitive function and freedom from frailty. The contribution of the proposed research will be to define the impact of modifiable individual behaviors and structural factors on trajectories of cardiac function in very late life, establish the role of a novel and targetable biologic pathway, and quantify the relation of these trajectories to functional and neurocognitive outcomes highly relevant to older adults. This contribution will be significant in enabling the identification of persons at high risk of progressive LV dysfunction in very late life when traditional risk factors perform poorly, and in determining the importance of a promising and targetable biologic pathway to preserve cardiac function – essential steps to decrease HF-associated morbidity and mortality. This research proposal is fundamentally innovative in: (1) focusing on longitudinal cardiac imaging in very late-life (75 to 91 years of age) when data is sparse but CVD burden is high; (2) interrogating a novel biological pathway (cellular senescence) potentially impacting late-life cardiac, physical, and neurocognitive function using serial high throughput proteomics integrated with genomic data; and (3) using innovative analytic approaches to identify trajectories, including Bayesian nonparametric trajectory mixture modeling. The project outcomes are expected to provide a novel understanding of the shared biologic pathways underlying preservation of cardiovascular, physical, and neurocognitive function in late life.

NIH Research Projects · FY 2025 · 2017-07

PROJECT SUMMARY/ABSTRACT There are significant obstacles facing promising young physicians who are considering careers as clinician- neuroscientists. Fostering the simultaneous development of research and clinical acumen during residency training requires an intentional effort on the part of a training program and its associated faculty mentors. The UT Southwestern Advancement of Neuroscience Research Careers (UT SWANS) program has been developed to provide focused, dedicated, purposeful, and combined clinical and research training, mentorship, and multidisciplinary resources to prepare clinicians-in-training for future careers as clinician-scientists. In the initial funding cycle, UT SWANS was successful with its first trainees and now has a growing pipeline of promising physician-neuroscientists. As detailed in our proposal, we have identified several definable opportunities to enhance our program with this resubmission. Furthermore, with the appointment in the past 2 – 3 years of NIH-funded departmental chairs, the UT Southwestern Departments of Neurology and Neurological Surgery have enhanced recruiting, mentoring, and academic training opportunities to take advantage of the unique academic and research-training resources available at UT Southwestern. By simultaneously offering outstanding clinical training at state-of-the-art facilities, a slate of 29 diverse, talented and invested research and career mentors, and a collaborative neuroscience community, UT SWANS is positioned to train clinician-neuroscientists who not only are capable of conducting outstanding clinically- relevant research, but also achieving balance between academic and clinical demands. Our goal is to provide a program that will catalyze the careers of clinician-neuroscientists who will be future leaders. Trainees will be prepared to identify high impact research questions, conduct rigorous research, and achieve independent funding. To achieve this overall goal, UT SWANS proposes to accomplish the following specific aims: (1) Recruit clinician-neuroscientist trainees of outstanding potential in neurology and neurosurgery. (2) Foster a culture of inquiry. (3) Cultivate and deliver outstanding research and career mentorship. (4) Provide comprehensive training in neuroscience research methodology, research rigor, and responsible conduct of research. (5) Ensure trainees conduct high-quality research. (6) Promote communication skills, including grant writing and speaking. Likelihood of success is enhanced by our mPI structure, which provides complementary and new scientific leadership from Drs. Louis (an accomplished neurologist-scientist and mentor) and Pouratian (an accomplished neurosurgeon-scientist and mentor). The program draws on a diverse faculty across multiple departments to provide broad opportunities to pursue research careers across numerous disciplines. Finally, UT Southwestern is well positioned as an institution to accelerate training opportunities for clinician-neuroscientists, having just completed a $1Billion drive to support the Peter O’Donnell Brain Institute, which has committed additional resources for innovative multidisciplinary neuroscience research and training.

NIH Research Projects · FY 2025 · 2017-04

Project Summary/Abstract (30 lines) MicroRNAs (miRNAs) constitute a large family of short, non-coding, regulatory RNAs that modulate protein expression. Abnormal miRNA levels are associated with many diseases, including developmental defects and various cancers. To generate functional miRNAs, primary transcripts (pri-miRNAs) generally need to be first cleaved by an RNaseIII, Drosha. This critical step of miRNA biogenesis needs to be controlled, in both accuracy and efficiency, to maintain proper gene regulation. The processing enzyme Drosha requires its partner protein, DGCR8, for protein stability and substrate specificity. A Drosha molecule binds homo-dimeric DGCR8 to carry out pri-miRNA processing, but higher-order complexes may also form because clustering of pri-miRNAs in the genome enhances processing. Our recent groundbreaking cryo-electron microscopy (cryo- EM) structures provide atomic models of the Microprocessor-pri-miRNA complex in action. Elucidating how the proteins are organized around the RNA stem-loop also revealed how each distal end (basal or apical) is independently recognized but also linked to each other via a molecular ruler connecting the detection modules. The proposed research builds on our previous successes, as the structural framework will enable us to gain novel fundamental insights into how the macromolecular recognition is accomplished. Our overall goal is to understand the recognition of pri-miRNAs by Microprocessor at the molecular level. We hypothesize that RNA structural features and context-dependent sequence preferences dictate the processing fate of each individual pri-miRNA. We will dissect how the recognition is accomplished at each of the basal and apical junctions of pri- miRNAs. We will also investigate how diverse RNA sequences and structures from different pri-miRNAs affect proper recognition at each junction, to reveal the plasticity of the multipart machinery. Our previous work has also left us poised to address urgent questions on how clustering of pri-miRNAs enhances processing by Microprocessor, which is crucial for our overall understanding of miRNA biogenesis and has profound implications for the interpretation of previous and future results in a wide variety of fields obtained by manipulation of miRNA genes. A better grasp of the core recognition mechanisms will help us explain unique targets such as clustered pri-miRNAs. Together, the proposed studies will provide a comprehensive understanding of processing and regulation of miRNAs, important regulators of gene expression. Our work on deciphering how structure affects RNA recognition is a fundamentally important question and likely be insightful for many processes involving structured RNAs beyond miRNAs.

NIH Research Projects · FY 2025 · 2016-09

Project Summary/Abstract The physiological underpinning of motor symptoms in Parkinson disease (PD) remains incompletely understood. We propose that the dynamic nature of basal ganglia thalamocortical (BGTC) network activity accounts for and is critical for understanding the dynamic symptomatology of PD and the pathophysiology of disease. We believe that the failure to focus on and investigate the non-stationarity of BGTC physiology and movement kinematics significantly contributes to inconsistency in published results and has impeded progress. We propose and investigate a novel model that accounts for the underexplored temporally dynamic cascade of physiological events occurring between nodes in the BGTC motor circuit. We hypothesize that transient exaggerations in network-level coupling that result in impaired information flow trigger pathophysiological and motor sequelae of PD, including rigidity and bradykinesia, allowing for and differentiating pathological and non-pathological synchrony. We hypothesize that the likelihood of pathological synchrony resulting in impaired information flow depends on the “movement” state, accounting for disproportionate difficulty with movement initiation in PD. We also hypothesize that treatment (dopaminergic and deep brain stimulation [DBS]) decreases the probability of a synchrony-triggered pathological cascade, with some common final changes in the network (e.g., cortical phase amplitude coupling) but with specific differences in physiological effects due to distinct sites of therapeutic action. We will build on prior success of investigating PD network physiology in patients undergoing DBS implantation surgery by simultaneously assessing population level activity from multiple BGTC nodes, including motor cortex, dorsal premotor cortex (to where pallidal-receiving thalamic regions project), subthalamic nucleus (STN), and globus pallidus (GPi, in separate patients), in relation to clinical symptoms and behavior. We now also integrate single unit physiology and synchronized dynamic tasks to test our model. In Aim 1, we will establish the dynamic relationship between network synchronization, local oscillations, and pathophysiologic sequelae under different therapeutic conditions, including STN and GPi DBS and dopaminergic therapy. We hypothesize an increased probability of synchrony leading to pathologic sequelae in the “off” state and test specific hypotheses about both common and distinct physiological effects of the different therapies, depending on site of action. In Aim 2, we hypothesize and aim to demonstrate that movement-related brain states affect sequelae of network synchrony both physiologically and behaviorally, differentially impacting movement initiation and ongoing activity. Finally, in Aim 3, we will distinguish normal and pathologic synchrony (across therapeutic and movement conditions) using a novel information theoretic frameowrk, with a focus on the impact of criticality, complexity matching, and impairments in information flow. This work will enhance the BGTC functional wiring diagram by defining the pathophysiologic significance of network synchrony in PD. Addressing this gap will facilitate therapeutic innovations, including identification of signals for adaptive DBS and to guide pharmacologic innovation.

NIH Research Projects · FY 2025 · 2016-09

Project Summary/Abstract (Overall) Adolescent idiopathic scoliosis (AIS) is a twisting condition of the spine and is the most common pediatric musculoskeletal disorder, affecting 3% of children worldwide. Children with AIS risk severe disfigurement, back pain, and physiologic dysfunction later in life. Girls requiring treatment for AIS outnumber boys by more than five-fold, for reasons that are unknown. Hospital charges for AIS surpass one billion dollars annually in the U.S. and are rising significantly faster than for other pediatric procedures. AIS is treated symptomatically rather than preventively because the underlying etiology has been poorly understood. Genetic contributions to AIS are significant, but few human susceptibility loci were identified prior to the beginning of this Program. The mechanisms driven by these loci were likewise largely unknown, as they mapped within non-coding genomic regions that were not easily interpreted. The AIS field also lacked appropriate animal models that enable mechanistic and therapeutic studies. To address these issues, we established an innovative collaborative approach combining three Projects to lead unbiased gene discovery in humans, modeling and gene discovery in zebrafish, and genomic analysis of postnatal spine development. Our program addressed six gaps in knowledge: (i) identity of the tissue and cellular origins of AIS; (ii) defining the true beginning of AIS disease pathogenesis; (iii) defining the genetic factors and genetic interactions underlying AIS; (iv) developing robust vertebrate systems to functionally validate, interpret, and model human genetic findings; (v) defining the molecular mechanisms controlling spinal development post-somitogenesis, and the correlation with AIS; (vi) defining the basis of sexual dimorphism in AIS. In the prior award cycle our Program significantly advanced each of these initiatives. Integrating data in humans and animal models, our data underscored cartilage as a functional tissue in AIS and specifically highlighted the extracellular matrix compartment, new paradigms in the field. Our Program discovered several new AIS genetic susceptibility loci in human and developed 73 new zebrafish models of spine deformity. Data from each Project also converged on the hypomorphic nature of AIS disease alleles, supporting multigenic inheritance. We defined the non-coding regulatory landscape of human and mouse AIS-related tissues, and discovered that knockout of one such regulator linked to female AIS in humans produces a female-biased phenotype in mouse. Here we propose a comprehensive plan to drive these discoveries forward to define AIS disease mechanisms using genetically targeted mouse and zebrafish models, to define cell-specific transcriptional, epigenetic, and signaling mechanisms underlying AIS, to continue identifying vertebrate models of spine deformity by forward genetic screens in mouse and zebrafish, and to discover new high-risk alleles contributing to AIS in patients refractory to treatment. We will also expand our investigations to address why AIS has a female bias, and to testing rationally selected drug therapies in vertebrate models. These studies will advance fundamental understanding of AIS, inform diagnosis and highlight potential therapeutic targets.

NIH Research Projects · FY 2025 · 2016-09

My lab seeks to understand the cell biophysical mechanisms regulating transmembrane signal transduction, i.e. signal transfer from ligands to receptors to downstream effectors. There is mounting evidence that cell surface receptors and associated proteins exhibit a high degree of dynamic organization at the plasma membrane (PM), which is critical for their ligand binding and signaling. However, many fundamental questions remain unanswered. Toward filling this gap in our knowledge, my lab will pursue two research directions over the next five years. The first direction will investigate how focal adhesions, the actin cortex, and interactions with integrins regulate the spatiotemporal organization and signaling of the endothelial cell receptor VEGFR2. VEGFR2 is the main promoter of angiogenesis (the formation of new blood vessels from existing vessels) in both normal physiology and disease. Thus there is great interest and need to understand the mechanisms that regulate its signaling. These studies will reveal mechanisms that underlie the activation of multiple pathways downstream of VEFR2, and that underlie the integration of multiple external signals at the level of the PM. As VEGFR2 belongs to the large family of receptor tyrosine kinases, our studies are expected to reveal general principles of the regulation and signaling of this important family of receptors. The second direction will focus on the novel organizational principle of liquid-liquid phase separation (LLPS) for proteins at the PM, using the transmembrane protein LAT as a model system. LAT is critical for the activation and immune function of T cells upon encountering an antigen presenting cell. Recent in vitro reconstitution work suggests that signaling clusters composed of LAT and its downstream effectors are formed through LLPS. The cellular environment is however much more complex than an in vitro reconstituted system. Thus we will investigate to what extent LAT microclusters at the PM of T cells are formed through LLPS, and the mechanisms that regulate LAT cluster composition. These studies will shed light on the role of LLPS for protein organization within the cellular environment in general. Both research directions – by their very nature – require probing molecular activities with high specificity and resolution in their native cellular environment. To achieve this, we will develop integrative approaches based on live-cell single-molecule, super-resolution and activity biosensor imaging, combined with cutting-edge mathematical and statistical analysis tools to extract quantitative information from the experiments and to multiplex the complementary information that the different imaging modalities provide. These analytical tools will be critical for our studies because single-cell and single-molecule microscopy often reveal molecular and cellular heterogeneity that is difficult to digest without such tools. We expect our novel analytical tools to be broadly applicable to other systems studied via similar experimental approaches. The knowledge gained from our studies will provide the building blocks for deepening our understanding of cell signaling from the single-molecule level to the systems level.

NIH Research Projects · FY 2024 · 2016-09

Project Summary: Urinary Stone Disease (USD) is an increasingly prevalent and highly recurrent condition associated with major morbidity at a rising cost to society. Thus, improved management can significantly reduce its health burden. Increasing fluid intake is recommended to all USD patients. However, knowledge gaps persist regarding the impact of fluid therapy in preventing USD recurrence including effectiveness of strategies to achieve and maintain a high urine volume, and whether such strategies reduce USD recurrence. The Prevention of Urinary Stones with Hydration (PUSH) study is a randomized clinical trial investigating the impact of increased fluid intake and increased urine output on the recurrence rate of USD in adults and children. In this study 1,642 participants will be randomized to a control or intervention arm. Participants in both arms receive a “smart water bottle”. The intervention arm involves an additional program of behavioral interventions, including financial incentives, structured problem solving, and low touch interventions designed to improve adherence to a prescribed fluid intake regimen. The primary endpoint is occurrence of a stone event during a two-year observation period. The PUSH study is in its third year, and due to multiple challenges to recruitment of study participants, follow-up of participants and data collection have not yet been completed. Additional time is needed to ensure study completion and to accomplish all study goals. Although ureteral stenting is routinely performed after urological procedures for USD to mitigate peri-operative complications, stents cause significant patient discomfort. The causal mechanisms are only partly understood. The STudy to Enhance uNderstanding of sTent-associated Symptoms (STENTS) is a prospective observational cohort study enrolling adolescents and adults undergoing ureteroscopic intervention for ureteral and/or renal stones. Participants undergo detailed symptom assessment using validated questionnaires, a psychosocial assessment, quantitative sensory testing for evaluation of pain sensitization, and detailed collection of clinical and operative data. Biospecimens (blood and urine) are being collected for future research. Recruitment to the STENTS study and follow-up of the participants are expected to be completed on time. However, additional time and resources are needed for analysis of collected study data. In Aim 1 of this application, the investigators will continue and complete participant enrollment for the PUSH study, continue biospecimen collection for the NIDDK Repository, analyze the data, and prepare and submit several planned manuscripts related to the study hypotheses. In Aim 2 of this application, the investigators will analyze the data from the STENTS studies, interpret findings, and disseminate findings through peer reviewed publications.

NIH Research Projects · FY 2025 · 2016-08

Overall Summary Particularly prevalent in Texas, renal cell carcinoma (RCC) is lethal when metastatic. To address this unmet medical need, the UTSW Kidney Cancer SPORE has developed a comprehensive therapeutic program in proven (targeted therapies and immunotherapy) and innovative (metabolism-directed) areas. Arguably, the most important driver of RCC is HIF2α. Discovered at UTSW, and regarded as undruggable, structural studies revealed a vulnerability that was exploited through a chemical screen leading to the founding of Peloton Therapeutics in the UTSW BioCenter and the development of PT2385 and PT2977. During the previous funding period, Project 1 investigators validated HIF2α as a target, identified putative biomarkers of dependency, executed a phase 1 trial, identified resistance mutations, and established HIF2α as a core dependency. Culminating the vertical collaboration and program success, Peloton was acquired by Merck, and PT2977 (also called belzutifan) gained FDA approval. During the next period, an innovative siRNA-based, second-generation inhibitor targeting both wild-type and resistant mutant HIF2α will be co-developed together with a ground- breaking imaging radiotracer enabling HIF2α evaluation in patients. Project 2 investigators exploit a profound link between RCC and metabolism. Using pioneering isotope-labeled nutrient infusions, Project 3 investigators established during years 1-5 that glutamine is a key nutrient fueling RCC growth in patients. In years 7-12, they will deploy the authenticated In Vivo Metabolism Lab to target glutamine bypass pathways, likely explaining the recent failure of glutaminase inhibitors. Finally, by leveraging Breakthrough Prize-recognized research at UTSW leading to a new innate immune system-activating drug, Project 3 investigators propose a paradigm shift in immunotherapy development involving the coordinated activation of the adaptive and innate arms (as it occurs physiologically). Together with previously commended development and career-enhancing programs, SPORE investigators are supported by four Cores. A forward-looking Administrative Core (Core A) serves as a hub. A Pathology Core (Core B) brings to bear one of the largest and most sophisticated RCC tumor banks and expertise supporting national efforts. A Data Analytics Core (Core C) assists with statistical support, bioinformatics, and data management with an avant-garde tool that automatically extracts information from the electronic medical record, self-updates, and links this information to experimental genomics and the tumor bank. An Imaging Core (Core D) delivers enabling technologies, including IND-holding innovative tracers, and unqualified expertise. Building upon the Simmons Comprehensive Cancer Center Kidney Cancer Program and its history of collaborative, interdisciplinary cancer research, SPORE Projects and Cores provide an engine of discovery, innovation, and translation supporting national and international efforts to advance patient care, research, and education.

NIH Research Projects · FY 2026 · 2016-08

Project Summary/Abstract To adapt to metabolic cues and survive stresses like nutrient starvation, cells store high- energy lipids in specialized organelles called lipid droplets (LDs) that emerge from the endoplasmic reticulum (ER), the metabolic center of cells. Recent studies reveal that LDs serve many roles in cell physiology, but how they are functionally ear-marked for specific tasks is unclear. Indeed, the ER network itself executes numerous cellular functions, but how this functional diversity is mechanistically achieved is unclear. The purpose of this grant is to dissect the molecular mechanisms by which LDs and the ER network achieve functional diversity at the sub-organelle level. Capitalizing on published and preliminary data, we find that LDs exhibit unique surface proteomes that provide functional specificity for LDs within single cells. Furthermore, we find that inter-organelle contact sites define ER sub-domains with specific roles in the compartmentalization of mevalonate metabolism. Here, I outline three directions that enable the mechanistic dissection of LD and ER functional compartmentalization: 1) In direction 1, we will leverage yeast genetics, biochemistry, and metabolic dissection to dissect how yeast ER-lysosome contacts (called nucleus-vacuole junctions, NVJs) serve as ER sub-domains and “metabolic platforms” that spatially compartmentalize mevalonate metabolism, and promote metabolic remodeling during glucose starvation. 2) In direction 2, we will capitalize on a large-scale screen to dissect how LDs are labeled with specific proteins to enable unique roles within cells. 3) In direction 3, we will utilize state-of-the-art cryo-FIB-SEM imaging to dissect how lipid phase transitions within LDs selectively remodel the LD surface proteome, and ultimately influence LD function and LD inter-organelle contact sites. Collectively, these directions will provide insights into new cellular organizational principles that enable to LDs and the ER network to achieve a functional division-of- labor. They will also characterize lipid phase transition properties of sterols, which promote disease pathologies in obesity, heart disease, and atherosclerosis.