Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2022-08

Tau is a microtubule-associated protein that converts from a healthy shape into beta-sheet rich amyloid structures which underlie pathology in Alzheimer’s disease and over 20 other related tauopathies. Recent cryo- EM structures of tau fibrils derived from different tauopathy patient samples reveal structural polymorphisms that link each tauopathy to distinct amyloid structures. The J-domain protein (JDP) molecular chaperone family play a central role in regulating tau function to mitigate amyloid assembly in disease. How the diverse family of JDP molecular chaperones discriminate binding different tau conformations remains poorly understood. My lab employed a CRISPR knock-out screen for factors that regulate tau aggregation and identified two JDPs, DnaJC7 and DnaJB6. In recently published worked, we revealed that DnaJC7 has enhanced specificity for natively folded wild-type tau compared to aggregation-prone mutants or pathogenic seeds. We hypothesize that the diverse JDP family encodes selectivity for different conformations of substrates. Our data suggests that novel mechanisms of JDP regulation of tau aggregation could be exploited as both a diagnostic and a therapeutic intervention. Several important questions remain. How do JDPs that bind to tau discriminate different tau conformations? Can JDPs modify tau pathology in vivo? In this proposal, we aim to define how a subset of JDPs recognize different conformations of tau linked to different tau pathologies in cells, in vitro and in vivo. We will first use biochemical and cellular approaches to understand how JPDs recognize different conformations of tau. We will also determine structures of tau in complex with DnaJC7 and DnaJB6. Finally, we will test the activity of DnaJC7 and DnaJB6 on the development of tau pathology in vivo. Our long-term goal is to develop therapies that can reduce amyloid deposition and slow neurodegeneration. This project is in alignment with the mission of the NIA to support biological and clinical research on aging.

NIH Research Projects · FY 2025 · 2022-08

Metastatic disease, or the spread of cancer cells from invasive primary tumor to distal organs through vasculature or lymphatics is responsible for majority of patient deaths and remains a clinical challenge. Not all invasive tumors are metastatic, therefore defining the determinants of metastatic competence and identifying cancer patients likely to develop metastasis or have residual disease is critical for clinical management of disease. Intratumor heterogeneity, inability to precisely isolate tumor cells from the invasive front and limited access to clinical follow- up data present a major challenge to identify metastatic competency determinants. Invasive intravascular growth is observed in approximately 15% of ccRCC patients as tumor thrombus. This invasive tumor extension can be accurately identified using cross sectional imaging including computerized tomography and magnetic resonance imaging scans and is valuable for tumor staging. We investigated resected invasive primary tumors with clearly defined extension into the vasculature to identify ccRCC invasion and metastasis determinants. Approximately fifty percent of ccRCC patients with intravascular tumor extension develop metastasis post nephrectomy and treatment and have poor survival outcome. Comprehensive transcriptomic analysis of metastatic and non-metastatic invasive ccRCC tumors with intravascular extension identified metastatic competence determinants. Our central hypothesis is that metastatic competence of invasive ccRCC tumors is dependent on PRRX1 driven vasculogenic mimicry and the ability of chemotactic cytokines to recruit CXCR2 positive infiltrating immune cells to the invasive front that promote dissemination and colonization to distal organs. Pharmacologic and conditional genetic manipulation of PRRX1 and CXCL1 ligands in experimental model systems resulted in attenuated metastasis. We will investigate the role of PRRX1 mediated vasculogenic mimicry in driving metastasis and assess the impact of TGF-β inhibitor on metastatic progression. We will also elucidate the role of CXCL1 in driving metastasis and assess the impact of CXCR2 antagonist on metastatic progression. We anticipate this project will decipher pathophysiologic mechanisms determining metastatic competency of invasive ccRCC and develop innovative therapies to disrupt metastatic competence, which in combination with current standard of care regimens will result in effective management of metastatic disease.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY With the alarming increase in the incidence of infections caused by antibiotic-resistance bacteria, there is an urgent need to identify new strategies to combat this emerging threat. The development, growth, and survival of all living organisms rely on coordinated gene expression. Central to gene expression is RNA polymerase (RNAP), a multi-subunit protein that transcribes genetic information from DNA to RNA in the complex and highly regulated process of transcription. Transcription has three major stages for creating a nascent RNA: initiation, elongation, and termination, each of which is controlled by protein transcription factors. RNAP is a proven drug target, but RNAP’s mechanistic features and how it is regulated by transcription factors remain poorly understood in pathogenic bacteria. My long-term goal is to understand the mechanisms of action of RNAP and key transcription factors involved in regulating RNAP initiation (CarD), elongation (NusG and NusA) and termination (Rho) in order to improve future antimicrobial development. In this proposed research, I will investigate the biochemical, structural, and genetic basis of the transcriptional machinery of Clostridioides difficile (C. diff.), a life-threatening gut pathogen that is resistant to multiple antibiotics. In Aim 1(K99 phase), I will investigate the functional relationship between two paralogs of the transcription regulator CarD and RNAP through in vitro and in vivo studies to test the hypothesis that the two CarD paralogs compete to bind and regulate RNAP, and the interplay of these factors is critical for coordinated control of transcription initiation in C. diff. In Aim 2 (K99/R00 phase), I will use genomic-scale mapping techniques and genetic assays to interrogate how Rho rewires gene expression by terminating transcription by RNAP. I will also design biochemistry assays to elucidate the mechanisms by which NusA and NusG, two universal elongation factors, modulate Rho-RNAP behavior. In Aim 3 (R00 phase), I will build an in vitro platform using the Broccoli fluorescent RNA aptamer to enable high-throughput screening of inhibitors of C. diff. RNAP. Virtual screening will be conducted to identify novel inhibitors based on our newly obtained cryo-EM structure. The proposed research in the K99 phase will mainly be conducted in the lab of Prof. Robert Landick at the University of Wisconsin-Madison. The key area that I will acquire additional research training is genome-scale mapping techniques and corresponding bioinformatics skills to analyze high-throughput datasets. I will also be guided by an advisory committee including collaborators Prof. Federico Rey (UW-Madison, an expert in microbiome-host interactions) and Prof. Elizabeth Campbell (The Rockefeller Univ., an expert in cryo-EM of RNAP and associated proteins), and consultant Prof. Joseph Sorg (Texas A&M Univ., an expert in C. diff genetics and physiology). I will also benefit from the facilities and abundant resources at UW-Madison. During the mentored phase of this award, I also plan to hone my skills in teaching, leadership and scientific communication, which will facilitate my transition to an independent research career.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Alcohol is the most commonly used drug in the United States and its use is on the rise. Alcohol abuse can result in alcoholic fatty liver disease, and even cirrhosis, as the majority of alcohol is metabolized by the liver during first pass metabolism. Alcohol is primarily metabolized in the liver through two enzymatic reactions (alcohol dehydrogenase and aldehyde dehydrogenase 2) that each reduce an NAD+ to NADH, contributing to a reduced redox state. Alcohol consumption reduces the redox state (greater NADH/ NAD+ ratio) which may impair β-oxidation and the TCA cycle. These factors may result in a reduction in fatty acid oxidation and the accumulation of lipids. Metabolic mechanisms in the liver may also use acetate as substrate to contribute to de novo lipogenesis, further contributing to alcoholic fatty liver disease. However, as much as 80% of the acetate produced during alcohol metabolism escapes the liver and can be used by peripheral tissues or organs such as the heart. The heart is also severely affected by alcohol abuse, which can lead to cardiac dysfunction and the development of various cardiovascular diseases, such as alcoholic cardiomyopathy. In the heart, acetate interferes with fatty acid oxidation, which results in lower ATP concentrations, as well as the accumulation of triglycerides. Acetate may impair the oxidation of free fatty acids (FFAs) as fuel, not through the inhibition of CPT via malonyl-CoA production, but by metabolic mechanisms outcompeting CPT for free CoAs therefore resulting in the limitation of FFAs entry into the mitochondria for oxidation. Administering acetate in in vitro (heart perfusions) and in vivo infusion experiments results in an energy deficit through a mechanism that has yet to be elucidated. It is possible that this energy deficit, and lipid accumulation, in the heart are contributing to cardiac dysfunction. The aim of this proposal is to examine these particular metabolic mechanisms and determine whether they are responsible for the alcohol induced energy deficit in the heart. This proposal will also determine whether these mechanisms are responsible for mediating alcohol induced cardiac dysfunction. A secondary aim is to determine, through the use of tracers, how chronic alcohol consumption changes overtime and how the liver and heart metabolize and use alcohol metabolites.

NIH Research Projects · FY 2025 · 2022-08

Hepatocellular carcinoma (HCC) is one of the fastest-growing cause of cancer death in the U.S. and it is projected to be the 3rd leading cause of cancer death in the U.S. by 2040 given the poor effectiveness of current HCC risk stratification and early detection strategies. Specifically, HCC screening is recommended in all patients with cirrhosis, despite annual HCC risk varying between 1-4%/year, highlighting a need for risk stratification biomarkers. HCC screening is performed using abdominal ultrasound and the serum biomarker alpha fetoprotein (AFP); however, this strategy misses over one-third of HCCs at an early stage and results in screening harms in many patients. The goal of our Clinical Validation Center for HCC (CVC-HCC) is to validate novel blood and imaging biomarkers in phase I-III studies to improve HCC risk stratification and early detection. Translation of HCC biomarkers to practice has been hampered by a dearth of high-quality sample sets including both stored blood and imaging. Existing sample sets also primarily include patients with cirrhosis from active viral hepatitis, with limited applicability to contemporary populations who primarily have cured viral hepatitis or non-viral causes of liver disease. Our CVC will create a contemporary resource with blood and imaging data to allow for rapid validation of promising biomarkers for HCC risk-stratification and early detection in phase I-III studies. A specific population in need of better biomarkers is patients with indeterminate liver nodules (ILNs) on diagnostic CT or MRI, which are observed in over one-fourth of patients undergoing HCC screening and have a high, yet variable, risk for developing into HCC (annual risk ~6-10%/year). Our group has validated a novel blood- based biomarker, PLSec, for risk stratification and a biomarker panel, GALAD, for early HCC detection in patients with cirrhosis and herein propose to perform a phase II-III biomarker study to evaluate them in patients with ILNs. Our team includes national leaders in HCC screening, imaging, and biomarker validation. We are leading efforts to evaluate HCC biomarkers including the EDRN-funded Hepatocellular Early Detection Strategy (HEDS) Study, NCI-funded Translational Liver Cancer (TLC) Consortium, and CPRIT-funded Texas HCC Consortium. We will leverage existing infrastructure across five health systems to create two novel resources not offered by the current sample sets including (1) a biorepository with both blood and imaging data from patients, with and without HCC, representing contemporary etiologies of liver disease for Phase II studies and (2) a prospective cohort of patients with ILNs to evaluate HCC risk stratification and early detection biomarkers in Phase III studies using a prospective-specimen-collection, retrospective-blinded-evaluation (PRoBE) design. We will work with the BCCs and DMCC to evaluate novel biomarkers, facilitating contributions to trans-network projects. Overall, our CVC- HCC will lead to significant advances in phase I-III validation of novel biomarkers for HCC risk stratification and early detection, areas of need that will facilitate development of well-designed phase IV clinical utility trials.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Protein ADP-ribosylation (ADPr) is a dynamic, NAD+-dependent post-translational modification. The mammalian poly(ADP-ribose) polymerase (PARP) proteins that catalyze ADPr target several chemically distinct amino acid side chain functionalities on hundreds of substrate proteins to mediate a multitude of orthogonal signal transduction pathways. Adding to this complexity is the potential for ADP-ribose polymer formation, a process wherein the PARP1/2 and TNKS1/2 enzymes elongate ADP-ribose chains from mono-ADPr sites. Highlighting the importance of poly-ADP-ribose in physiology and disease are: (i) the expanding clinical utility of PARP1/2 inhibitors to treat DNA repair-deficient cancers, and (ii) TNKS1/2 function in Wnt/b-catenin signaling and dysfunction in developmental diseases including Cherubism. Aberrant ADPr activity has also been reported as an underlying cause of cardiovascular and neurogenerative diseases, and these findings have inspired intense efforts to elucidate PARP substrate profiles, determine PARP regulatory mechanisms, and develop PARP isoform-specific inhibitors. However, given the liberal deployment of ADPr in cellular signaling and its topologically complex chemical nature, our understanding of how specific mono- and poly-ADPr sites impact protein function and elicit distinct biological activities has lagged behind. The proposed work aims to fill this knowledge gap by developing novel approaches to reconstitute ADPr-mediated signaling events in highly controlled biochemical and cellular environments. We recently developed a chemoenzymatic strategy to install serine ADPr onto peptides and proteins with full control over modification site and ADP-ribose chain length. Using this technology, we identified critical molecular determinants of DNA damage-induced chromatin remodeling and uncovered specialized functions for nucleosome serine poly-ADPr. We are now in a unique position to build upon our technologies and address fundamental questions in PARP biology. We will explore mechanisms that govern poly-ADPr activity and investigate how different modification sites and accompanying polymer lengths encode for specific biochemical outputs throughout the cell. Such information may guide more effective strategies to identify and treat diseases that rely on dysfunctional ADPr activity.

NIH Research Projects · FY 2026 · 2022-08

PROJECT ABSTRACT: The objective of this NIA K23 proposal is to support my continued scientific growth towards becoming an independent translational, interventional neuropsychologist. Alzheimer’s clinical syndrome (ACS) involves amnestic mild cognitive impairment (aMCI), a stage preceding dementia, and has multiple risk factors. Traumatic brain injury (TBI) is one significant risk factor that remains poorly understood. An earlier onset of ACS has been linked to a TBI history, and my group published one of the first theoretical mechanistic models that posited biological changes involved in Alzheimer’s disease and related dementias (ADRD) may be increased from TBI. While moderate and severe TBI have a well-established link to ACS, it is unclear if mild TBI (mTBI), the most common TBI type, has a similar relationship. More information is needed to determine if biological changes in ACS, and possibly ADRD, might relate to mTBI. Such information can be generated from biomarker probing. The recent advent of high definition transcranial direct current stimulation (HD-tDCS) and blood-derived biomarker tools provide sophisticated new methods to probe biomarkers, specifically neural circuit integrity and neuronal injury/inflammation. To accomplish my career development and research goals, we have created a comprehensive training plan to develop new skills to probe biomarkers of neural circuit integrity and neuronal injury/inflammation to inform if biological changes in ACS relate to a history of mTBI. Through the K23, I will gain new knowledge about biological changes in ADRD and TBI, different fluid-based biomarker approaches, multiple noninvasive brain stimulation methods, translational research, leadership/ governance, and scientific networking. Training will include mentorship, didactic coursework, hands-on experiences, and the scientific study. The mentoring team is an interdisciplinary group of leaders with expertise in ADRD, TBI, noninvasive brain stimulation, biomarkers, neurobehavioral research design, and academic career development. The scientific study’s overarching hypothesis is that the risk for aMCI associated with mTBI will manifest as reduced HD-tDCS-measured neural circuit integrity and elevated blood biomarkers of neuronal injury/inflammation. The proposal will leverage the UT Southwestern Alzheimer Disease Center to enroll adults with aMCI. Aim 1 will determine if neural circuit integrity involved in verbal episodic memory in aMCI is reduced based on a history of mTBI by using a within-subjects design to apply three HD-tDCS conditions. Aim 2 will determine if key blood-derived markers of neuronal injury/inflammation in aMCI are elevated based on having an mTBI history. The K23 proposal will provide essential data and first-authored publications to prepare my first NIA R01, enable me to independently design clinical/translational research that incorporates biomarkers, and position me to achieve my overall career goal of informing the biological mechanisms linking onset of ACS, and potentially ADRD, to a history of TBI. Once these goals have been achieved, I will utilize the new insights to explore potential therapeutic interventions later in my career.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Heart failure is a leading cause of morbidity and mortality around the world, and the leading cause of heart failure with reduced ejection fraction (HFrEF) is coronary artery disease. The mitogen-activated protein kinases (MAPK) are an essential signal transduction cascade that play a central role in both cell growth and cell death. The MEK1-ERK1/2 branch of the MAPK pathway has been shown to promote both physiologic and pathologic growth in the heart, as well as protect against apoptotic cell death after ischemia/reperfusion (I/R) injury. We have previously demonstrated that the transcription factor ETS2, a member of E26 transformation-specific sequence (ETS)-domain family, is phosphorylated and activated by Erk1/2 upon hypertrophic stimulation. Going forward, our preliminary data reveal that cardiomyocyte- specific loss of ETS2 results in increased susceptibility to ischemic injury in both I/R and permanent ligation (myocardial infarction) models of heart failure. Connexin43 (Cx43), the predominant gap junction channel- forming protein in cardiomyocytes, has been suggested to play a role in both ischemic damage and ischemic preconditioning. Our preliminary data show that ETS2 activates Cx43 transcription and that Cx43 is downregulated in the absence of ETS2. We will test the hypothesis that the ERK1/2/ETS2 pathway protects against I/R injury in part through the upregulation of Cx43. Aim 1: To determine the role of the ERK1/2-ETS2 pathway in I/R injury. Our preliminary data suggest a model in which ETS2 protects against I/R injury. We will confirm and extend this using loss- and gain-of- function approaches in vivo and in vitro. We will track the timing of ETS2 activation by ERK and the response of each acutely and in long-term remodeling. Aim 2: To determine the impact of ETS2 on Cx43 expression and function in I/R injury. Our data suggest that Cx43 plays a protective role in ischemic injury. Our preliminary data also suggest that ETS2 is a direct transcriptional regulator of Cx43 gene expression. We will confirm and extend these findings using both loss- and gain-of-function approaches. We will also determine the role of ETS2 in Cx43-mediated cardioprotection in IPC. Aim 3: To determine the downstream targets and interactors of ETS2 under conditions of cardiac stress. We will profile genome-wide cardiac gene expression using RNAseq and ChIPseq to determine ETS2 downstream gene targets in both acute and long-term I/R injury. We will use immunoprecipitation and mass spectrometry to unveil novel protein interactions.

NIH Research Projects · FY 2025 · 2022-08

Nutrient digestion and absorption are essential functions of the gastrointestinal (GI) tract. By the time the intestinal contents reach the colon, the organ is mostly responsible for water and electrolyte reabsorption. Nonetheless, through the metabolic activities of the colonic microbiota, non-digestible luminal contents are fermented resulting in the flux of many small metabolites that are utilized by both microbial organisms as well as by the host. In fact, it is now estimated that bacterial metabolism contributes ~6-10% of the calories that reach the host. Hence, mechanisms to sense and respond to the metabolic flux originating in the colon would seem essential to metabolic homeostasis. In this regard, the GI epithelium contains enteroendocrine cells (EECs), which produce a variety of hormones that help coordinate GI physiology, as well as metabolic responses in a variety of distant organs. Considering that the location of most digestive and absorptive processes is the small intestine, it is intriguing that the colon produces several hormones that control host metabolism and appetite (e.g., Glp-1, Glp-2, Insl5). Using a mouse model of colonic EEC deficiency (EECCol) we have uncovered that these cells are critical to host metabolic homeostasis. We find that these mice develop obesity, which is mostly due to hyperphagia. Moreover, it is associated with changes in intestinal microbiota (dysbiosis). We hypothesize that the spatial distribution of colonic EECs reflects the significant caloric flux derived from microbial metabolism of non-digestible nutrients. The purpose of this project is to evaluate how colonic EECs modulate host metabolism, with the underlying hypothesis that they act as proximal sensors of metabolic flux originating in the colon. To address this goal, we propose the following aims: Aim 1: How does dysbiosis contribute to the metabolic phenotype resulting from colonic EEC deficiency? Hyperphagia and obesity in EECCol mice can be prevented by treating their dysbiosis. Here we will test the hypothesis that bacterially derived metabolites are responsible for modulating appetite and body weight. Aim 2: How is colonic EEC deficiency able to affect microbiota composition? In this aim will test the hypothesis that host digestion and absorption of nutrients from the intestinal lumen is impacted by colonic EECs and that this in turn influences carbon sources and microbiota composition. Overall, the pursuit of this project will provide novel insights into the role of colonic EECs in the regulation of host metabolism and will fill key gaps in our understanding of how the intestinal microbiome regulates metabolic homeostasis more broadly.

NIH Research Projects · FY 2025 · 2022-08

Impact of Intensive Treatment of Systolic Blood Pressure on Brain Perfusion, Amyloid and Tau in Older Adults (IPAT-study) Project Summary Recently, the NIA-AA research framework has defined AD as a biological construct of abnormal accumulation of Aβ and tau proteins in the brain. Similarly, the importance of cerebrovascular contributions to AD pathogenesis is now well recognized. Hypertension is the leading cause of cerebrovascular disease; >70% of adults aged 65 or older have hypertension. The SPRINT trial showed that intensive treatment of hypertension reduced risk of cognitive impairment or dementia. However, the underlying mechanisms are unclear. Hypertension and the associated arterial stiffening compromise regional cerebral blood flow (CBF), reduce brain white matter integrity, and impact brain amyloid and tau clearance via the brain glymphatic system. Our studies also showed that high blood pressure and central arterial stiffness are associated positively with brain Aβ burden measured with PET and that the amplitude of low frequency fluctuations of blood-oxygen-level- dependent signal measured with rs-fMRI (BOLD ALFF) is correlated negatively brain amyloid burden in older adults, suggesting its role in brain Aβ regulation. The overarching goal of this project is to determine whether intensive lowering of systolic blood pressure (SBP) to a target of <120 mmHg, compared with <140 mmHg, reduces brain amyloid and tau in older adults who are at high risk of dementia. Furthermore, we will determine the impact of BP lowering on CBF, arterial stiffness, BOLD ALFF, white matter hyperintensity (WMH), brain network connectivity, and neurocognitive function, as well as the relationships of these changes with brain amyloid and tau. We will enroll 180 older adults age 60 to 80 years who have hypertension (SBP≥130 mmHg), FH of dementia, and/or subjective memory complaints. Participants will be randomized into the intensive treatment (SBP<120 mmHg) or usual care (SBP<140 mmHg) arms and followed for 2 years to accomplish the following specific aims: 1) To determine the effects of intensive SBP lowering on brain amyloid, tau, and neurocognitive function. Hypotheses: Intensive SBP lowering, when compared with usual care, reduces the progression of brain Aβ and tau deposition; changes in tau are correlated with neurocognitive function. 2) To determine the effects of intensive SBP lowering on CBF, central arterial stiffness, and BOLD ALFF. Hypotheses: Intensive SBP lowering reduces central arterial stiffness and increases regional CBF and BOLD ALFF; changes in CBF, arterial stiffness, and BOLD ALFF are correlated with brain Aβ and tau. 3) To determine the effects of intensive SBP lowering on brain WMH, white matter microstructural integrity, and neural network connectivity. Hypotheses: Intensive SBP lowering reduces the progression of brain WMH, improves white matter microstructural integrity and brain network connectivity which are correlated with changes in brain Aβ and tau. The new knowledge obtained will provide mechanistic insights into the relationship between hypertension, cerebrovascular function, and AD pathophysiology which is potentially important for development of multidomain strategies for dementia prevention and treatment.

NIH Research Projects · FY 2026 · 2022-08

Overview. Electrical signal transmission between cells underlies almost all physiological processes in human, from heartbeat, to learning and memory. Neurotransmitter receptors are organized in clusters at micron-sized inter-cellular machineries, namely synapses, throughout our nervous system to enable signal transmission. Decades of intense research have characterized the working mechanism of most individual receptors. However, due to technical difficulties, the clusters of receptors remain enigmatic – neither the structure, nor the functional significance is known. In this proposal, my lab will use state-of-the-art methods and a reconstitution system to systematically characterize the architecture of synaptic glycine receptors, which is the last major neurotransmitter receptors whose architecture remained elusive before our work. We will also develop novel technologies for quantitative characterization of higher-order assemblies of glycine receptors, and other proteins in the 2D setting of lipid membranes. Following is a brief description of the proposed work. Glycine receptors and its clustering with gephyrin scaffold. Glycine receptor (GlyR) belongs to the Cys-loop family of pentameric ligand-gated ion channels. GlyRs in adult tissue are heteromeric receptors composed of both the α and β subunits, which form clusters with scaffold protein gephyrin at synapses through a specific interaction between the β subunit and gephyrin. Disfunction of GlyR signaling is the major cause of the rare congenital disease hyperekplexia and related to chronical neurological pain and autism spectrum disorders. The architecture of heteromeric GlyRs and how they form clusters with gephyrin is very limited – even the α:β subunit stoichiometry has been under debate for decades. We have discovered an unexpected subunit composition of GlyR, explaining the unique function of heteromeric GlyRs and clearing long-lasting confusion. We will use this established platform to characterize all major types of GlyRs and how they interact with gephyrin. Develop novel technologies for characterizing protein clusters in lipid membrane. My lab will develop a novel correlated raised-total internal refection fluorescence microscopy (TIRF) and electrophysiology reconstitution system to characterize ion channel/receptor clusters. This system has the high signal/noise ratio and single- molecule sensitivity as traditional TIRF, and allow complex electrophysiological experiments to be performed simultaneously with imaging. We will learn how clusters form, how they are regulated, and whether clustering gives rise to functional effects and regulate physiological activities. The knowledge gained here will guide the reconstitution of functional clusters for structural characterization using cryo-EM single particle and/or tomography methods. These new methods will allow quantitative characterization of the spatial organization and functional significance of clustering, as well as the mechanisms underlying assembly and functionality. My lab will apply these novel methods to investigate GlyR-gephyrin clusters as the first example in class, and open new research directions in analyzing other receptor clusters found ubiquitously in biology for signal transmission.

NIH Research Projects · FY 2026 · 2022-08

Project Summary/Abstract Abnormal chromosomes are hallmark features of human diseases and genetic disorders. Cancer genome sequencing has uncovered a complex class of localized genomic rearrangements, known as chromothripsis, that arises from the catastrophic fragmentation of individual chromosomes. Chromothripsis is initiated by mitotic cell division errors resulting in the formation of micronuclei, aberrant nuclear structures that transiently encapsulate mis-segregated chromosomes outside of the nucleus. Micronuclei serve as hotspots for the accumulation of extensive DNA double-strand breaks (DSBs) by restricting DNA damage to a confined region of the genome. A detailed mechanistic understanding of chromothripsis, however, has been limited by inherent challenges in monitoring micronucleated chromosomes for more than one cell cycle. We recently bypassed this limitation by developing a platform that enables the controlled induction of chromosome-specific micronuclei in human cells. By reconstructing the cascade of events resulting in chromothripsis, we found that damaged micronuclear DNAs are susceptible to fragmentation upon premature chromosome condensation triggered by mitotic entry. These fragments undergo error-prone DSB repair during the subsequent cell cycle to generate diverse chromosomal rearrangements that are identical to those found in cancers and genomic disorders. Moreover, we identified that short DNA fragments entrapped in the cytoplasm can activate a cell-autonomous immune response. Despite this knowledge, we currently have a limited mechanistic understanding of the consequences of chromosome fragmentation. For example, it remains unclear how pulverized fragments from micronuclei re-incorporate into daughter cell genomes during mitosis and become reassembled by one or more DSB repair mechanisms throughout interphase. Additionally, it is unknown whether chromosome fragmentation can elicit a non-cell autonomous response. Here we outline our research program over the next five years aimed at understanding the fate of micronucleated chromosomes across different phases of the cell cycle and its mutagenic consequences on genome integrity. Using time-lapse light-sheet microscopy, we will interrogate the spatiotemporal dynamics of chromosome fragmentation, movement, and reassembly during mitosis and interphase. This will be achieved by engineering a CRISPR-based labeling strategy to visualize micronucleated chromosomes undergoing chromothripsis in living cells. Next, we will identify how the DNA damage response and distinct DSB repair pathways orchestrate the reassembly of chromosome fragments to shape the genomic rearrangement landscape of mitotic errors. Lastly, we will investigate how chromosome fragments residing in the cytoplasm can elicit inter-cellular consequences with neighboring cells in the environment, including the lateral exchange of genetic material. Altogether, these studies aim to define fundamental principles governing the intrinsic and extrinsic fate of micronuclei in initiating catastrophic genomic alterations. The proposed research will fill a critical gap in our understanding of how cell cycle errors can rapidly drive somatic mutagenesis.

NIH Research Projects · FY 2025 · 2022-08

Electroconvulsive therapy (ECT) is one of the most effective antidepressant non-invasive brain stimulation therapies for adults with major depression. However, a number of patients fail to respond despite adequate trials, and while clinically beneficial, ECT can produce adverse cognitive effects including amnesia, executive dysfunction, and verbal dysfluency. Previous single- and multi-site ECT-imaging investigations have been limited by insufficient sample size and/or non-standardization of methodology. Therefore, in answer to NIMH Strategic Objective 3.2 “Develop strategies for tailoring existing interventions to optimize outcomes,” our investigative teams have conducted clinical studies to develop standardized methods for acute ECT course administration, antidepressant and cognitive measures for phenotyping, optimal neuroimaging protocols and E-field modeling, and sophisticated analytic models to integrate and interpret the antidepressant-response and cognitive- impairment biomarkers. In this prospective study we propose the first investigation integrating multiple units of analysis including clinical and cognitive phenotyping, whole-brain neuroimaging, EEG, and E-field modeling to establish the mechanisms underlying ECT-induced antidepressant response (response biomarkers) and cognitive adverse effects (safety biomarkers), as well as to find the “sweet spot” of ECT dosing for optimal antidepressant benefit and cognitive safety. Adult patients with major depressive disorder (n = 230) will receive a standardized acute ECT course, complete clinical and cognitive measures and undergo structural and functional MRI at three time points (baseline, after ECT #6, and following treatment completion) and one-month naturalistic follow-up. All MRI data will be processed and harmonized identically at a central imaging core to ensure uniformity. We have three primary aims: 1) Determine the relationships between E-field strength, ictal power, and biomarkers; 2) Determine the relationships between E-field strength, biomarkers, and antidepressant outcomes; and 3) Determine the relationships between E-field strength, biomarkers, and cognitive outcomes. An exploratory aim will contrast antidepressant-response and cognitive-impairment biomarkers identified in the current proposal with magnetic seizure therapy and healthy comparison subjects. The overarching hypothesis of this investigation is that the E-field variability will explain antidepressant and cognitive outcomes. Public Health Significance: Successful completion of this project will verify the optimal ECT dose (the “sweet spot”) of 112 V/m within the right hippocampus which can then inform precision and individualization of ECT amplitude with “E-field informed ECT”. The standardized algorithms for E-field modeling can be generalized and widely disseminated. This proposal will result in a paradigm shift from “trial and error” approaches of ECT parameter selection to individualized, precision dosing to improve patient outcomes.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY This study is designed to answer one of the fundamental gaps in knowledge in the resuscitation of preterm infants at birth: What is the optimal target oxygen saturation (SpO2) range that increases survival without long- term morbidities? Oxygen (O2) is routinely used for the stabilization of preterm infants in the delivery room (DR), but its use is linked with mortality and several morbidities including bronchopulmonary dysplasia (BPD). To balance the need to give sufficient O2 to correct hypoxia and avoid excess O2, the neonatal resuscitation program (NRP) recommends initiating preterm resuscitation with low (≤ 30%) inspired O2 concentration (FiO2) and subsequent titration to achieve a specified target SpO2 range. These SpO2 targets are based on approximated 50th percentile SpO2 (Sat50) observed in healthy term infants. However, the optimal SpO2 targets remain undefined in the preterm infants. Recent data suggest that the inability to achieve SpO2 of 80% by five minutes is associated with intraventricular hemorrhage (IVH) and mortality. These studies raise concern that current SpO2 targets (Sat50) may be too low resulting in persistence of high pulmonary vascular resistance, respiratory failure and mortality. Preliminary data from my NICHD K23 funded pilot randomized controlled trial (RCT) of 75 preterm infants <31 weeks gestational age (GA) showed that infants randomized to 75th percentile SpO2 goals (Sat75) had a lower incidence of SpO2 <80% at five minutes in the DR compared to infants randomized to 50th percentile SpO2 goals (Sat50). In addition, Sat75 infants had less oxidative stress at one hour after birth, needed less respiratory support on admission, had less pulmonary hypertension and had higher survival without BPD. We hypothesize that delivery room resuscitation of preterm infants < 31 weeks GA with Sat75 targets compared to Sat50 targets will increase survival without BPD by 36 weeks’ postmenstrual age (PMA). We plan to conduct a multicenter RCT of Sat75 versus Sat50 powered for survival without BPD. We will randomize 772 infants, 230/7- 306/7 weeks’ GA, to Sat75 (intervention) or Sat50 (control). Except for the SpO2 targets, all resuscitations will follow NRP guidelines including an initial FiO2 of 0.3. In Aim 1, we will determine whether targeting Sat75 compared to Sat50 increases survival without BPD. In addition, we will compare the rates of other major morbidities such as IVH. In Aim 2, we will determine whether targeting Sat75 compared to Sat50 increases survival without neurodevelopmental impairment at 2 years of age. In Aim 3, we will determine whether targeting Sat75 compared to Sat50 decreases oxidative stress. We will conduct a sub-study of 220 infants enrolled from a single site to measure 8-iso-PGF2α and 8-OHdG in cord blood and blood samples collected at 1 and 24 hours after birth. The new understanding gained from this trial has the potential to modify neonatal resuscitation practice and improve neonatal outcomes worldwide. This will be the first clinical trial powered for survival without BPD to evaluate competing DR SpO2 goals and addresses a critical knowledge gap recognized by NRP and NICHD.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Acute myeloid leukemia (AML) is one of the most aggressive hematologic malignancies in adults, yet decades- old chemotherapies remain the standard of care and few targeted therapies exist owing to its molecular and clinical heterogeneity. An emerging hallmark of AML development is the epigenetic silencing of LINE1 retrotransposons which is required to maintain AML self-renewal, differentiation blockade, and genomic stability. Aberrant reactivation of LINE1 retrotransposons selectively impairs propagation of human and mouse AML cells without affecting normal hematopoiesis; however, it remains elusive how LINE1 activity inhibits myeloid leukemogenesis. Aberrant retrotransposon reactivation by cancer-targeting epigenetic inhibitors such as DNA hypomethylating agents (DMA) produces a type I interferon (IFN)-mediated ‘viral mimicry’ response in various cancer types, including leukemias, resulting in cell cycle arrest and apoptosis. We hypothesize that LINE1 retrotransposons contribute to the development of myeloid leukemia by modulating type I interferon signaling mediated by cGAS and/or RIG-I-like Receptor (RLR) sensing of LINE1 gene products. This proposal will establish the functional role of LINE1-mediated IFN signaling in myeloid leukemogenesis and determine the mechanisms by which LINE1 activates innate immune ‘viral mimicry’ pathways in AML cells. The Specific Aims of this proposal intend to 1) establish the functional role of LINE1-mediated interferon signaling in myeloid leukemogenesis and progression; and 2) identify the molecular sensors of LINE1-mediated interferon activation in AML. Ectopic LINE1 overexpression and CRISPR activation of endogenous LINE1s in human AML cells will determine whether LINE1 expression induces type I IFNs and impacts cell proliferation, myeloid differentiation, and/or apoptosis. Combining a conditional LINE1 activation transgenic mouse with the established MLL-AF9 retroviral leukemia model will determine whether activation of LINE1s induces hematopoietic-specific type I IFN to impair AML initiation and/or maintenance in vivo. Moreover, genetic ablation of cGAS, RIG-I, or MDA5 nucleic acid sensors individually or in combination in human AML cells and their corresponding knockout mouse models will determine how loss of DNA- and/or RNA-sensing pathways affects LINE1-induced inflammation and AML pathogenesis in vivo. Altogether, these stringent genetic studies will provide direct evidence to establish the functional role of LINE1-mediated IFN signaling in myeloid leukemia. Addressing these outstanding knowledge gaps will be critical to inform whether and how modulation of the retrotransposon-innate immunity crosstalk may be leveraged as a new mechanism-based therapeutic strategy to selectively eradicate AML cells.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY / ABSTRACT Phosphatidylinositol 4,5-bisphosphate (PIP2)-Ca2+ signaling is activated following stimulation of growth factor receptors, antigen receptors, and G protein-coupled receptors by ligands including neurotransmitters and histamine. The activation of phospholipase C triggers hydrolysis of the PIP2 lipid at the plasma membrane (PM) to generate diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 subsequently releases Ca2+ stored in the endoplasmic reticulum (ER) to activate cytosolic Ca2+ effectors for downstream signaling events such as secretion, proliferation, and migration. The consumed PM PIP2 and ER Ca2+ must be quickly restored to sustain signaling responses and to maintain cellular homeostasis. This homeostatic regulation of PIP2-Ca2+ signaling requires transport of phosphatidylinositol (PI) from the ER to the PM for PIP2 resynthesis and store-operated Ca2+ entry (SOCE) that activates Ca2+ influx to refill the ER Ca2+ store. SOCE has been studied extensively; however, the mechanisms underlying PIP2 replenishment during PIP2-Ca2+ signaling are not well understood. Recently, we and others discovered the lipid transfer protein Nir2 that localizes at ER-PM junctions, where the ER is in contact with the PM, to mediate PM PIP2 replenishment. The objective of this proposal is to define the mechanisms regulating PIP2 replenishment by Nir2 at ER-PM junctions during receptor-induced Ca2+ signaling. Our preliminary studies identified Nir1, an Nir protein lacking a lipid transfer protein domain, as a binding partner and positive regulator of Nir2 at ER-PM junctions. In addition, our recent data revealed that the membrane- shaping hairpin sequence present in all extended synaptotagmins (E-Syts) is important for regulating PIP2 replenishment at ER-PM junctions. Moreover, we found that conversion of DAG into phosphatidic acid (PA) by DAG kinases (DGKs) is crucial for Nir2 localization at ER-PM junctions. Among the ten DGKs in mammalian cells, the epsilon-isoform of DGK (DGKε) is recently shown to localize at ER-PM junctions. Our central hypothesis is that PM PIP2 replenishment during receptor-induced Ca2+ signaling is mediated by the oligomers of Nir1 and Nir2 formed following PA production from DAG by DGKε at ER-PM junctions shaped by E-Syts. We propose three specific aims to test our central hypothesis using biochemical analysis and advanced imaging techniques to determine the mechanisms by which Nir1, E-Syts and DGKε regulate PIP2 replenishment. We expect that successfully completion of the proposed studies will establish the molecular and cellular mechanisms regulating PIP2 replenishment following hydrolysis induced by receptor stimulation. Restoring PM PIP2 is critical to sustain Ca2+ signaling and maintain PM PIP2 levels critical to membrane trafficking, cytoskeletal dynamics, and ion transport in receptor-stimulated cells. Mutations in Nir1 are linked to retinal dystrophy and patients with mutations in DGKε suffer from renal failure. Our proposed studies may provide knowledge for developing therapeutic strategies to treat patients affected by these diseases.

NIH Research Projects · FY 2026 · 2022-07

Summary Aging represents a major risk factor for a broad range of diseases and declines in tissue homeostasis and function. This is particularly true in the female reproductive system where the aging of stored oocytes has been directly linked with an increased incidence of miscarriages and birth defects. Our long-term goal is to identify and characterize the factors that contribute to reproductive aging. In mammals, eggs can be stored months, years, or decades, making the analysis of reproductive aging slow and experimentally difficult. Here, we seek to build upon previous efforts to establish the Drosophila ovary as a powerful system with which to study reproductive aging. Interestingly, the decline in egg quality has been correlated with lower levels of mRNA translation across species, from flies to humans. Despite this common defect, we know surprisingly little about the mechanisms responsible for this reduction of mRNA translation capacity within stored eggs. Here, we propose to use state of the art genetic manipulation and biochemical analysis to systematically characterize how the machinery required for mRNA translation changes with maternal age and during egg storage in Drosophila. Moreover, we seek to genetically test whether manipulating ribosome levels and translation initiation/elongation rates prolongs the quality of stored eggs. We have established an operational pipeline for conducting all the experiments outlined under this proposal and seek to take advantage of a number of innovative tools and techniques that have been adopted by our group. Under Aim 1, we will use complementary molecular and biochemical approaches to comprehensively characterize the extent to which protein synthesis and ribosome levels changes in the ovaries of aging females and in eggs stored over two weeks. We will also use biochemical and innovative reporter based assays to evaluate whether translation fidelity declines with age. In aim 2, we will test the extent to which increasing or decreasing ribosome levels and translation initiation and elongation rates improves the quality of stored eggs. Under aim 3, we will characterize how the ribosome oxygenase NO66 influences egg quality. We believe this comprehensive analysis of in vivo oocytes during the course of aging will provide key insights into why the quality of eggs declines with age and will reveal new molecular targets for the development of therapies designed to improve and extend reproduction. Given our focus on the role ribosomes play in this process, we believe our work will broadly impact the study of tissue homeostasis and regeneration in aging organisms.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY The prevalence of obesity and its complications, including diabetes and non-alcoholic fatty liver disease (NAFLD) are a significant health care crisis. These complication impact liver metabolism by dysregulating gluconeogenesis, lipid synthesis, mitochondrial function, and fat oxidation. NAFLD is improved by lifestyle interventions. Ironically, physical activity, exercise, and aerobic capacity affect many of these pathways similarly, but profoundly lower liver fat independent of weight loss. Hence, aerobic exercise is commonly prescribed therapy for NAFLD. One route by which obesity or exercise influence regulation of liver metabolism may be by signals through the sympathetic nervous system. The overarching goal of this 5-year research career development plan is to facilitate my transition from a technically focused researcher to a fully independent academic scientist investigating the in vivo physiology of disease. This will be accomplished by training in disciplines of physiology, neurophysiology, and exercise that will be used to identify mechanisms by which hepatic sympathetic nervous signaling controls liver metabolic flux during obesity and interventions. Elevated basal sympathetic signaling is thought to occur through the α1b adrenergic receptor (AR), which is highly expressed in mouse liver (including hepatocytes) and has been suggested to stimulate hepatic glucose production, breakdown of glycogen, gluconeogenesis, tricarboxylic acid (TCA) cycle and ketogenesis. This project focuses on understanding how hepatic α1b-AR contributes to dysregulated hepatic gluconeogenesis and fat oxidation during obesity and NAFLD (Aim 1), and the degree to which liver α1b-AR mediates beneficial effects of acute (Aim 2) or chronic exercise (Aim 3) as treatments of NAFLD. Using targeted metabolomics and stable isotope infusions, I will quantitatively evaluate how AR signaling regulates liver metabolism. The findings will advance our knowledge of how metabolism is altered by complications of obesity and provide a novel training platform for the recipient.

NIH Research Projects · FY 2025 · 2022-07

Hepatocellular carcinoma (HCC) is the fastest rising cause of cancer-related death in the U.S. and a leading cause of death in patients with cirrhosis. Epidemiologic data demonstrate that certain populations, including men and older individuals, have consistently higher HCC incidence and mortality across diverse settings and time periods. Despite this well-established pattern, the underlying biologic and behavioral contributors to these differences remain unclear – particularly among patients with cirrhosis, the primary at-risk population in the U.S. This project applies a multilevel conceptual framework to identify and evaluate specific biological and behavioral factors that may influence variation in HCC incidence and outcomes. This study leverages three large multicenter cohorts of patients with cirrhosis and HCC, each with robust clinical data, patient-reported outcomes, and stored serum samples. The central hypothesis that variation in HCC risk is more strongly associated with biologic mechanisms, while variation in survival may be more closely related to behavioral and social factors, will be investigated through the following Specific Aims: 1) Estimate the association between biologic characteristics and HCC risk in >4,000 patients with cirrhosis; 2) Quantify differences in HCC survival across patient subgroups; and 3) Evaluate how biologic and behavioral factors contribute to differences in HCC survival. Findings from these project aims will be triangulated to inform future strategies aimed at improving health for patients with cirrhosis and reducing the burden of HCC. The PI is a clinical researcher and hepatologist at UT Southwestern with a long-term goal of improving liver cancer outcomes through a deeper understanding of the individual-level risk factors that drive both risk and disease progression. The proposed training plan integrates advanced methods in clinical epidemiology, behavioral science, and analysis of biologic data and is supported by a multidisciplinary team of nationally recognized mentors. This project has significant

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT The molecular basis of gastric cancer (GC) health disparities that Hispanic/Latino (Hs/L) patients face is an understudied and unmet public health issue. Compared to non-Hispanic Whites, Hs/L patients with GC are younger, have twice the disease incidence, and are more likely to develop the more aggressive form of the disease called diffuse GC. The molecular causes for these disparities are unknown since Hs/L patients have not been included in previous GC studies. The investigators recently completed the first integrated genomic analysis of Hs/L GC patients and found that 7 of 43 (16%) Hs/L patients with diffuse GC carried germline CDH1 variants. Germline CDH1 variants that are pathogenic cause hereditary diffuse GC syndrome (HDGC), which confers up to an 80% lifetime risk of developing diffuse GC, often at a young age. Thus, Hs/L patients may have a higher rate of HDGC, which would help explain the unique clinicopathologic characteristics seen in these patients since HDGC is thought to cause <1% of GC. There is a critical need to define HDGC prevalence in Hs/L patients as the syndrome may be a cause of GC health disparities. However, determination of the true rate of HDGC is hampered by two obstacles: 1) current tools are unable to determine if most CDH1 variants are pathogenic or benign (3 of 7 variants identified in Hs/L patients had uncertain function), and 2) the penetrance of CDH1 variants is unpredictable. While obesity is associated with being diagnosed with GC, preliminary work by the investigators shows that obesity may also influence disease penetrance by inducing earlier disease onset. The objective of this proposal is to identify molecular mechanisms for GC health disparities. The hypothesis is that a higher prevalence of HDGC and effect modification by obesity contribute to worse outcomes in Hs/L patients with GC compared to White patients. An innovative translational project that blends clinical epidemiology and experimental biology will be performed to pursue the following aims. Aim 1 will determine the prevalence of CDH1 variants and how they associate with genetic ancestry and lifestyle/environmental exposures in Hs/L and White patients with diffuse GC. Hs/L patients will be enrolled from around the world. Aim 2 will functionally assess whether discovered CDH1 variants confer pathogenic behavior using both in vitro and in vivo systems. Aim 3 will ascertain the effect of obesity on CDH1 variant penetrance. The project's innovations are: 1) accounting for the heterogeneity of the Hs/L population, 2) using novel functional methods to ascertain the pathogenicity of CDH1 variants, and 3) studying obesity as a modifier of GC penetrance. The impact of the expected results would be the identification of the first known molecular mechanism for GC health disparities. Determining that obesity augments CDH1 penetrance would open novel lines of inquiry into gene-environment interactions that drive GC formation. The results could be clinically actionable by informing genetic testing criteria and lifestyle recommendations that enable the prevention or early detection of diffuse GC in Hs/Ls. Finally, 60% of HDGC cases have no known cause; the proposed methods can be applied to test other potential HDGC causes.

NIH Research Projects · FY 2025 · 2022-07

A Multifaceted Radiomics Model to Predict Cervical Lymph Node Metastasis for Involved Nodal Radiation Therapy PROJECT SUMMARY The majority of disease sites treated with radiation therapy (RT) no longer receive elective/prophylactic RT to clinically-negative areas, including lung, pancreas, and lymphoma. These disease sites now employ involved nodal radiotherapy (INRT), focusing on involved lymphadenopathy. However, in head and neck cancer (HNC), we still target the same lymph node regions as conventional 2D radiotherapy, despite our ability to tailor the radiotherapy volume and dose to specific areas using intensity modulated radiation therapy (IMRT). This approach leads to excessive acute and long-term toxicities for HNC patients after RT. Therefore, INRT is highly desirable for HNC. In INRT, one particular challenge during gross tumor volume (GTV) and clinical target volume (CTV) delineation is the identification of malignant lymphadenopathy. While some lymph nodes (LNs) are obviously malignant based on standard imaging modalities, there is often uncertainty about whether a LN is malignant and requires targeting. Treating benign nodes as malignant may cause a significantly higher risk of late complications, such as xerostomia and dysphagia. On the other hand, missing occult lymphadenopathy will lead to regional recurrence. The goal of this project is to develop, optimize, and test a multifaceted predictive model with both high sensitivity and specificity for LN metastasis classification to maximize the efficacy and minimize the toxicity of INRT for HNC. The proposed multifaced model presents a flexible framework and considers multiple aspects of a predictive model, including: 1) Evaluation criteria used in model training (multi- objective); 2) Different sources of information (multi-modality); and 3) Classifiers used for model construction (multi-classifier). By designing a multi-objective function, we will consider sensitivity and specificity simultaneously during model training and optimization. Instead of blindly combining features extracted from different modalities and empirically choosing one preferred classifier, the information extracted by modality- specific classifiers will be combined optimally through a reliable classifier fusion (RCF) strategy. We will develop a prospective registry database to train the multi-classifier, multi-objective and multi-modality (MCOM) model through prospectively collecting clinical characteristics and images of HNC patients who will undergo surgery at UTSW with pathology-confirmed LN metastasis status. The model will be validated on an independent UTSW patient cohort and patients who underwent outside imaging but operated at UTSW. The specific aims of the project are: 1) Develop and validate a multi-classifier, multi-objective and multi-modality (MCOM) LN metastasis prediction model for HNC patients. 2) Conduct a randomized phase II clinical trial to evaluate the efficacy and utility of INRT versus conventional radiotherapy for HNC using the MCOM model. Successful completion of this project will result in the development and validation of a strategy that can identify malignant LNs in HNC with high sensitivity and specificity, which will lead to improved outcomes for HNC patients who receive INRT.

NIH Research Projects · FY 2025 · 2022-07

Project abstract X-ray crystallography and cryoEM single particle reconstruction (cryoEM SPR) generate uniquely detailed structural information that is used to: (1) understand cellular processes at the molecular level, (2) explain and validate results obtain by other biochemical, biophysical and cell biology methods, and also (3) guide drug design studies. All these applications are highly relevant to the NIH mission. The proposal aims to advance data analysis methods for X-ray and cryoEM diffraction and cryoEM SPR so that reliable and informative structural models can be obtained from micro- and nanocrystals with both techniques as well as from single molecules (particles) with electron microscopy. The PI aims to expand X-ray crystallography and cryoEM SPR methods to new areas by highly hierarchical application of data mining and dimensionality reduction methods. The data richness generated by recent changes in hardware enables deep exploration of much more elaborate, non-random start algorithms that have better convergence than the random start methods that are frequently used in computational approaches. In diffraction methods, one frequently needs to combine data from multiple crystals for successful structure solution. However, optimal averaging should only consider data that represent the same structural source of diffraction patterns, so there is a fundamental need to segregate individual samples into distinct groups that are internally isomorphous. In traditional approaches, complex non-isomorphism patterns result in combinatorial complexity of data analysis in the presence of incompleteness and low signal-to-noise for individually contributing datasets. The PI will develop methods addressing this long-standing unsolved problem, with the methods having potential to also advance the analysis of biologically relevant structural variability that manifests as non- isomorphism in experimental date. In cryoEM diffraction, data analysis does not yet produce reliable structural results consistently, de novo structure solution is limited to a small number of projects where direct methods can be used, and for small molecules, determination of absolute configuration remains a challenge. The PI will develop and implement experimental and computational solutions to advance modeling of systematic effects encountered in electron diffraction and to expand phasing approaches in electron crystallography to address these outstanding problems. The PI will also work on developing estimators of bias magnitude and debiasing procedures to expand cryoEM SPR so that much smaller particles can be modelled reliably. Finally, the PI will develop approaches relying on comparative genomics so that structural models can be built and validated at very low resolution that are currently outside of the reach for molecular interpretation. All research will rely on the strong expertise of the PI in selected areas.

NIH Research Projects · FY 2025 · 2022-07

Cell fate is controlled by master regulators, as is also the case in the nervous system. Pax6 is a highly conserved transcription factor consisting of two DNA-binding domains and a transcriptional activation domain. It is broadly expressed in neural stem/progenitor cells and plays pivotal roles in neuronal specification, migration, and axonal projections. Through a series of in vivo screens, we discovered that PAX6 is also capable of inducing new neurons from genetically traced glial cells in the adult mouse spinal cord. Based on such exciting data, we hypothesize that PAX6-induced adult neurogenesis from resident glial cells may represent an alternative strategy for neural regeneration. The major goal of this project is to characterize the PAX6-induced adult neurogenesis process and determine its functional role following traumatic neural injuries. We propose three specific aims to accomplish this goal. First, we will employ genetic lineage tracing and genome-wide deep sequencing to understand how PAX6 induces new neurons from resident glial cells in the adult mouse spinal cord. Secondly, we will employ recombinant virus-mediated transsynaptic tracing to delineate the network connections of PAX6- induced new neurons. And thirdly, we will use chemogenetics to tease out the functional role of PAX6-induced new neurons on behavior in adult mice with traumatic injury.

NIH Research Projects · FY 2026 · 2022-07

The goal of the UTSW NORC is to enhance research efforts in nutrition and obesity by facilitating interdisciplinary interactions to speed the translation of basic scientific discoveries into clinically relevant interventions in humans that improve public health. We have assembled a highly interactive and collaborative interdisciplinary team of investigators at UT Southwestern that harnesses expertise from multiple disciplines, with a goal of defining the behavioral, metabolic, genetic, and molecular mechanisms contributing to obesity and obesity-induced diseases. The strength of our faculty and the support of our administration provide the UTSW NORC with the expertise, infrastructure, and ability to develop new methods to study the effects of macronutrients on metabolism, new insights into the pathophysiology of obesity, and to develop new approaches for the treatment of obesity and obesity-associated metabolic co-morbidities. The UTSW NORC now brings together 204 investigators at UT Southwestern from 28 different departments at UT Southwestern to address critical questions in nutrition, obesity, and obesity-induced diseases. To facilitate interdisciplinary research, our NORC has created the following thematically related Teams: Team 1: Central Regulation of Energy Metabolism; Team 2: Adipocyte Biology and Energy Metabolism; Team 3: Obesity-induced Peripheral Organ Disease; and Team 4: Nutrition, Obesity, and Cancer. Within each team, we have integrated scientists from a wide spectrum of disciplines to address the biochemical, metabolic, and clinical consequences of obesity and related diseases. In addition, each Team has a mixture of senior, mid-level, and junior scientists. The Pilot & Feasibility program supports four new investigators/year who will also be assigned to one of the four research Teams. To support NORC investigators, we have established four cores and a Clinical Element to provide state-of-the art services that exceed the capabilities, availability and/or budgets of individual investigators. The Animal Phenotyping/Metabolism Core provides an extensive array of blood metabolites and hormones, 50+ metabolic cages, multiple imaging modalities to characterize fat distribution, and hyperinsulinemic-euglycemic clamps. The Lipid Mass Spectrometry Core provides comprehensive quantification of lipids. The Quantitative Metabolism and Imaging Core provides flux measurements of metabolic pathways in vivo and 1H imaging and spectroscopy to characterize skeletal muscle and liver lipid content in humans. The Genetics, Single Cell Sequencing, and RNA seq core provides exome sequencing and quantitative gene expression determinations and analysis. All cores provide services for both animal and human research studies. Thus, the UTSW NORC provides an important mechanism for a large group of productive investigators to coalesce and focus their research efforts related to nutrition and obesity to accelerate the translation of basic scientific discoveries into clinically relevant interventions in humans that improve public health.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Approximately 1.5 million of the 44 million Blacks in the United States are carriers of the valine-to-isoleucine substitution at position 122 (V122I) in the transthyretin (TTR) protein. Virtually exclusive to Blacks, this is the most common cause of hereditary cardiac amyloidosis (hATTR-CA) worldwide. hATTR-CA leads to worsening heart failure (HF) and premature death. Fortunately, new therapies that stabilize TTR improve morbidity and mortality in hATTR-CA, especially when prescribed early in the disease.(5) However, hATTR-CA is often diagnosed at an advanced stage and conventional diagnostic tools lack diagnostic specificity to detect early disease. Recent work from the author demonstrated that young V122I TTR carriers had indirect imaging and biomarker evidence of cardiac amyloid infiltration. Thus, the overall objectives of this proposal are to determine the presence of subclinical hATTR-CA and to identify biomarkers that indicate amyloid progression in V122I TTR carriers. The central hypothesis of this proposal is that hATTR-CA has a long latency period that will be detected through subclinical amyloidosis imaging and biomarker phenotyping. The central hypothesis will be tested by pursuing 2 specific aims: Aim 1) determine the association of V122I TTR carrier status with CMRI evidence of amyloid infiltration; Sub-aim 1) determine the association of V122I TTR carrier status with cardiac reserve; Aim 2) determine the association between amyloid-specific biomarkers and V122I TTR carrier status; and Sub-aim 2) determine the association of amyloid-specific biomarkers with imaging-based parameters and evaluate their diagnostic utility for identifying subclinical hATTR-CA. In Aim 1, CMRI will be used to compare metrics associated with cardiac amyloid infiltration between a cohort of V122I TTR carriers without HF formed by cascade genetic testing and age-, sex-, and race-matched non-carrier controls. For Sub-Aim 1, a sub-sample of carriers and non- carrier controls enrolled in Aim 1 will undergo novel exercise CMRI to measure and compare cardiac systolic and diastolic reserve. Aim 2 involves measuring and comparing amyloid-specific biomarkers in V122I TTR carriers without HF with samples matched non-carriers (both from Aim 1) and individuals with symptomatic V122I hATTR-CA from our clinical sites. These biomarkers detect and quantify different processes of TTR amyloidogenesis and include circulating TTR, retinol binding protein 4, TTR kinetic stability, and misfolded TTR oligomers. Sub-aim 2 will establish the role of these biomarkers to detect imaging evidence of subclinical hATTR- CA disease. The research proposed in this application is innovative, in the applicant's opinion, because it will enroll a large population of V122I TTR carriers without HF through cascade genetic testing that will model clinical care. Then, it will employ detailed, advanced imaging techniques with tissue characterization and novel biomarkers that directly quantify processes of hATTR-CA disease progression to identify subclinical hATTR-CA. Identifying evidence of cardiac amyloid progression prior to hATTR-CA disease onset will change how we think about this disease and justify future research in screening and treatment strategies to prevent hATTR-CA.