Ut Southwestern Medical Center

NIH Research Projects · FY 2024 · 2024-09

Project Summary Metastasis is the leading cause of death for cancer patients. Oxidative stress, characterized by excessive exposure to reactive oxygen species (ROS), kills most metastasizing cancer cells. How highly-metastatic clones manage to overcome both cell-intrinsic and extrinsic oxidative insults to colonize distant organs is poorly understood. The precise subcellular circuits enabling tumor adaptation to oxidative stress, and whether they could be exploited for therapy, have remained elusive. We hypothesize that organellar antioxidant pathways provide adaptive mechanisms essential for cancers to efficiently metastasize. A major impediment to testing this hypothesis has been the lack of high-resolution and versatile tools to study ROS in vivo. To address this major technological gap and address which subcellular redox circuits are necessary or sufficient for highly metastatic tumors to progress, we will develop and apply tools with exquisite spatiotemporal resolution in vivo. These tools include an optogenetic protein that produces localized ROS in tumor subcellular compartments, gene therapy strategies in mice that will pioneer the manipulation of tissue- extrinsic ROS in mouse tissues, and tumor organelle purification strategies coupled to mass spectrometry analyses in primary and metastatic tumors. With these tools in hand, this proposal aims to answer three key questions: Can the subcellular burden of ROS be exploited to hinder metastasis? Are there specific organelle-based nodes that enhance tumor antioxidant capacity for metastasis? Are extracellular ROS in the colonized target organ a major metastasis limitation? By integrating in vivo optogenetic modulation of ROS, high-resolution metabolomics, functional genetics and metastatic cancer models, our work will uncover targetable, ROS-mediated bottlenecks of metastasis at subcellular resolution. The tools and techniques developed in this proposal have the potential to revolutionize our ability to study ROS and oxidative stress both in vitro and in vivo, and be broadly applied to any disease impacted by ROS and oxidative stress. As such, my lab’s work has the power to reshape our understanding of these processes not only in cancer, but across a wide range of diseases, which may pave the way for new therapeutic strategies and improved patient outcomes.

NIH Research Projects · FY 2025 · 2024-09

Within the United States, 500,000+ individuals are enduring the long-term consequences of severe burn injuries covering 20% or more of their body surface area, with upwards to 11,000 individuals experiencing such an injury per year. These burn injuries severely compromise body temperature regulation, owing to permanent impairments of the primary heat-dissipating mechanisms, namely profoundly blunted skin blood flow and sweating in the injured skin. Furthermore, well-healed burn survivors exhibit greater all-cause mortality; more hospitalization days for “circulatory diseases” including heart disease, heart failure, and cerebrovascular disease; and have elevated incidences of diabetes, hypertension, coronary artery disease, and stroke. For more than 20 years, the PI has investigated the adverse consequences of severe burn injuries while focusing on two primary goals: a) providing important information to the burn survivor and their caregivers, targeted at improving the quality of life for burn survivors, and b) provide research-verified findings directed at reducing the elevated morbidity and mortality risk in well-healed burn survivors. Consistent with those goals, the objective of this MIRA application is to extend our discoveries, and associated understanding, of the cardiovascular and thermoregulatory consequences of a severe burn injury in humans and to pursue avenues to mitigate any such adverse consequences. Though specific aims are not permitted in MIRA applications, examples of research directions that could be pursued include quantifying cardiovascular function/dysfunction very early after a severe burn injury (e.g., within the first ~48 hours post-burn), at discharge from the hospital post-burn, and months after discharge; assessments of cardiac and renal stress due to physical activity in warm to hot environmental conditions in well-healed burn survivors while exploring avenues to mitigate any adverse responses; and perhaps identify the limits that a severe burn injury places on well-healed burn survivors’ thermoregulatory and cardiovascular capabilities to withstand adverse conditions associated with heat waves while also exploring approaches to mitigate such adverse responses. These research efforts will provide valuable information that will culminate in the reduction in otherwise heightened morbidity and mortality burden of burn survivors, along with associated improvements in cardiometabolic health, a greater ability to work and perform functions of daily living, improved quality of life, and enhanced independence as they age.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Living cells have a complex and often precise organization in space and time. Determining the three- dimensional structure of proteins and other biomolecules, as well as understanding how they form functional networks in vivo, is a major goal of modern biology. Answering these questions is paramount to understanding both the normal functions of proteins, as well as their dysfunctions. Cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) are powerful imaging tools that enable visualization and structural determination of native macromolecular complexes in vitro and in situ. Researchers use cryo-EM to resolve isolated (macro)molecules at near-atomic or atomic resolution, whereas cryo-electron tomography can visualize macromolecules and organelles inside unperturbed cells with molecular to near-atomic resolution. Together, cryo-EM and cryo-ET have the potential to reveal a more comprehensive and detailed (atomic-level) picture of the spatiotemporal organization and inner workings of cells. However, to fully realize the potential of cryo-EM/ET imaging techniques, we need new tools and approaches that can address outstanding technical limitations, such as radiation damage of frozen-hydrated biological specimens and the localization of specific molecules in cryo-tomograms. Therefore, we will develop a new sample preparation strategy that can reduce electron-induced radiolysis of frozen-hydrated specimens, thereby improving the resolution of cryo-EM/ET images and/or the speed of structure determination (Aim 1). Additionally, we plan to develop a cloneable, hyper-bubbling protein tag that would allow the precise localization of target proteins in otherwise noisy, and difficult-to-parse, cryo-tomograms (Aim 2). We will then apply these new tools to biological model systems (i.e. rapidly frozen and cryo-FIB milled E. coli and yeast cells) as proof of principle. The successful fulfillment of our research aims will further enhance the revelatory power of cryo-EM/ET techniques and illuminate the complex and dynamic relationship between molecular structure and function.

NIH Research Projects · FY 2025 · 2024-09

Nocturnin (NOCT) is a NADP(H) phosphatase with highly rhythmic expression peaking in the early dark phase (ZT12) in most tissues in mice. Noct-/- (Noct-KO) mice exhibit a range of tissue-specific phenotypes, but notably are resistant to diet-induced obesity and hepatic steatosis on a high-fat diet. Despite this protection from obesity and fat accumulation in the liver, these mice do not eat less and are not more active, but instead have alterations in energy expenditure that are not well understood. Given the widespread role of NADP(H) as an essential cofactor in numerous metabolic reactions, it is likely that NOCT has distinct tissue-specific effects, making it difficult to understand NOCT’s role in metabolism using a global KO model. Therefore, this proposal will use adipose-, liver-, muscle-, and intestine-specific conditional Noct-KO (cKO) mice, generated via the Cre- loxP system, to study NOCT’s role in metabolism. Aim 1 will determine if loss of NOCT in one tissue is responsible for the body weight phenotype. In this aim, both male and female cKO and control mice will be subjected to a high-fat diet for twenty weeks. Body weight and food intake will be recorded on a weekly basis, energy expenditure will be measured using metabolic cages and body composition measurements will be taken to determine differences in fat and lean mass. At the end of the study, plasma, adipose, liver, muscle, and intestinal tissues will be collected from each cKO line and their respective controls at two time points (ZT2 and ZT14) further analyses including measurement of plasma lipoproteins and plasma adipokine levels. Histology will also be done to define any gross morphological differences caused by loss of NOCT. Aim 2 will examine the gene expression changes in global Noct-KO and cKO mice. We will determine how the transcriptome of each of the four tissues change over the circadian day in the global Noct-KO when fed HFD for 20 weeks and will compare this to the transcriptome of each of the four individual tissues in the cKOs to assess the relative contribution of local NOCT activity on gene expression. In Aim 3 we will examine the molecular mechanisms that result in the protection of hepatic steatosis in the global Noct-KO and will determine whether this liver-specific phenotype is the result of loss of NOCT in the liver or requires contributions from other tissues. The experiments outlined in this proposal will define how tissue-specific loss of NOCT, and thus how tissue-specific increases in NADP(H) levels, impact metabolism. More specifically, this proposal will aid in understanding how loss of NOCT protects against diet-induced obesity and hepatic steatosis, which, in turn, can aid in the development of treatments for obesity and obesity-related diseases.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Mechanical loading of bones is important for healthy hematopoiesis and immune function. The bone marrow is sensitive to loading-induced changes in interstitial flow and shear stress. The Leptin receptor- expressing (LepR+) stromal cells in the bone marrow support both hematopoiesis and osteogenesis. Load- bearing exercise promotes the maintenance of lymphoid progenitors in the bone marrow by promoting signaling of the mechanosensitive ion channel Piezo1 in a subset of these cells: peri-arteriolar Osteolectin expressing (Oln+) cells. The hypothesis of this proposal is that another mechanically regulated ion channel, TREK1, contributes to the regulation of hematopoiesis and osteogenesis in the bone marrow. Trek1, an outwardly rectifying potassium channel that is activated by mechanical stretch and pressure, is expressed by most peri-arteriolar Oln+LepR+ cells and by a subset of other LepR+ cells. No one has investigated whether TREK1 regulates hematopoiesis or osteogenesis under physiological conditions. In Aim 1, I propose to test whether Trek1 expression in peri-arteriolar Oln+LepR+ cells and peri-sinusoidal Oln-LepR+ cells regulates hematopoiesis in the bone marrow. In Aim 2, I propose to test whether Trek1 expression in peri-arteriolar Oln+LepR+ cells and peri-sinusoidal Oln-LepR+ cells regulates osteogenesis in the bone marrow and whether this is modulated by mechanical loading. In Aim 3, I will test whether Trek1 expression defines a biologically or spatially distinct subset of LepR+ stromal cells in the bone marrow. I will test how the deficiency of Trek1 affects hematopoietic stem/progenitor cell frequency, osteogenesis, and bone marrow reconstitution capacity. Outcomes of the proposed studies will indicate whether Trek1 modulates hematopoiesis or osteogenesis in response to mechanical loading and whether Trek1-expressing cells perform distinct functions in the bone marrow niche.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY______________________________________________________________________ One-third of heart transplant recipients will develop acute rejection of their new heart within the first post- transplant year. These episodes can cause worsening heart failure, acceleration of chronic rejection, and decrease post-transplant survival. Although immunosuppression decreases the risk of rejection, its long-term use is associated with infection, cancer, and worsening kidney disease, all of which may limit the lifespan of a patient after transplant. The ability to safely minimize immunosuppression without increasing the risk of rejection would improve heart transplant recipients' outcomes. Calculating the risk of rejection for a given patient remains a clinical challenge, as age, sex, and immunologic mismatch between the donor and recipient are incomplete predictors of risk. While several non-invasive tests are available to diagnose rejection after transplant, there is not currently an assay that can be used before transplant to quantify whether a recipient is more prone to infection or rejection. If we could identify heart transplant candidates at low risk for rejection, we could safely minimize immunosuppression and its toxicities without increasing the risk of graft loss. The objective of the proposed study is to leverage a large, well-phenotyped cohort with existing biospecimens and multi-omic technologies to identify novel pre-transplant biomarkers associated with clinically significant rejection within the first post-transplant year. First, we will perform proteomic profiling to validate three biomarkers (FGF-2, SPRTY-2, IRAK-1) we identified in preliminary studies as associated with rejection. We will then expand our profiling on the Olink platform to include additional panels of proteins reporting on innate and adaptive immune activation. We will test whether biomarkers provide incremental predictive utility, beyond a set of prespecified clinical variables. Next, we propose to perform bulk RNA sequencing on peripheral blood mononuclear cells prospectively collected before transplant & compare differential gene expression of transcripts related to the protein biomarkers identified between recipients with and without rejection. We will also perform whole transcriptomic profiling and pathway analysis to identify relevant biological processes reporting on rejection risk. Finally, we will leverage novel machine learning approaches to identify an integrated omics signature of rejection risk. The results of the current study will support the submission of future grants to prospectively evaluate the use of these pre-transplant biomarkers in the clinical care of heart transplant recipients.

NIH Research Projects · FY 2025 · 2024-09

Obesity remains a major health problem in US and causes metabolic complications such as diabetes, dyslipidemia and insulin resistance. Similar complications also occur in patients with autoimmune lipodystrophies characterized by almost complete (acquired generalized lipodystrophy or AGL) or partial (acquired partial lipodystrophy, APL) loss of body fat. Recently, circulating autoantibody against perilipin-1 (PLIN1) has been implicated in the pathogenesis of AGL. However, nearly two-thirds of AGL patients do not have PLIN1 autoantibodies suggesting other adipocyte antigens may be involved in the autoimmune response. Furthermore, the pathogenesis of APL remains unknown. Thus, the first two aims of this proposal are to identify additional autoantibodies against adipocyte expressed proteins that cause AGL or APL and to determine their function in adipocyte biology. We will use two state-of-the-art complementary techniques, a. Phage ImmunoPrecipitation Sequencing (PhIP-Seq) Assay and b. Human Proteome Microarray (HuProtTM version 4 Chip) to identify the serum autoantibodies in AGL and APL patients. Our recent data from a mouse model of human autoimmune polyglandular syndrome type 1)(Aire-/- mice) reveal circulating PLIN1 autoantibodies and loss of both subcutaneous and visceral fat due to inflammatory lesions in adipose tissue. Therefore, the third aim of our proposal is to determine underlying autoimmune mechanisms involved in loss of tolerance to perilipin-1 in Aire-/- mice. Lastly, we will determine pathogenicity of novel autoantibodies discovered in patients with AGL and APL against adipocyte expressed proteins by infusing them into mice and evaluating loss of body fat, development of insulin resistance and metabolic derangements. These studies will unravel autoimmune mechanisms involved in causation of lipodystrophy, and insulin resistance and its associated morbidities. This new knowledge may provide targets for developing novel drugs for treating diabetes, dyslipidemias and hepatic steatosis.

NIH Research Projects · FY 2025 · 2024-09

Project Summary It is estimated that fixing problems related to preventable errors costs the U.S. healthcare system $400 billion annually. In the field of surgery, the most basic element of quality care begins in the operating room (OR), and >50% of preventable errors that lead to patient harm happen there. The suboptimal performance of OR teams often lead to adverse events such as wrong site surgery, foreign object retention, and delays that greatly increase morbidity, mortality, and costs. Though using a surgical safety checklist can prevent errors and reduce patient harm, measurement of improve critical intraoperative processes has traditionally required putting observers inside the OR to record and analyze team performance. Since this process is labor-intensive and costly, it has been limited to a few large academic centers conducting funded research in quality improvement. The operating room Black Box® (ORBB) is an innovative technological and analytical platform, that synchronously captures the performance of the operating surgeon and the OR team while simultaneously recording patients’ vitals and postoperative outcomes. The ORBB uses machine learning to analyze performance and outcomes, making the program scalable and unlocking the potential for widespread monitoring and improvement of intraoperative team performance. However, large-scale adoption of the ORBB is limited by a lack of training programs that incorporate the data into an actionable form and by the absence of high-quality data on how to implement the ORBB in diverse hospitals. We hypothesize that addressing these barriers will lead to significant improvement in outcomes for surgical patients and propose three specific aims to test these hypotheses. In aim 1, a high fidelity immersive multi-player virtual simulator will be developed for training the OR teams in performing the surgical safety checklist. In aim 2, the validity and effectiveness of the virtual simulator in improving the quality of safety will be studied using the ORBB data. In aim 3, a study of ORBB implementation will be conducted to identify barriers and facilitators to adoption of the ORBB across the US healthcare system.

NIH Research Projects · FY 2025 · 2024-09

Abstract Aging is the time-dependent functional decline responsible for increased susceptibility to chronic disease, frailty, and disability. Aging is a substantial public health concern, and new therapeutic interventions intended to mitigate its effects are needed. While preclinical evidence supports the crucial role of mTOR inhibitors in slowing aging processes, the lack of pharmacokinetic (PK) and pharmacodynamic (PD) data in older adults presents a substantial challenge in designing clinical trials. Sirolimus and everolimus are well-studied mTOR inhibitors commonly used in transplantation. However, both age and severe illness can significantly alter drug PK, and there is currently limited data in older adults without underlying, confounding illnesses. Additionally, there is a lack of data to inform the selection of PD biomarkers that optimally demonstrate improvement in age-related processes. Without well-defined PK characterization and identification of robust PD biomarkers, future clinical trials will lack the statistical power needed to establish evidence of effectiveness. To lay the foundation for future studies, we assembled a multidisciplinary team of clinical pharmacologists and epidemiologists, as well as experts in geriatrics, gerontology, and immunosuppression. The primary goal of this research is to precisely estimate fundamental PK/PD parameters of sirolimus and everolimus in older adults without confounding illness. We will first validate measurement techniques and quantify the in vitro exposure-response relationship for two different biomarkers: S6K activity and mitochondrial function (Aim 1). We will then conduct a clinical study to characterize the PK/PD of sirolimus and everolimus in older adults (Aim 2). Lastly, the biomarkers will be evaluated for their ability to demonstrate the magnitude of treatment response over an intermediate term follow-up (Aim 3). We anticipate that our research will address the existing knowledge gaps related to mTOR inhibitor PK/PD, leading to a transformative shift in the field of aging research.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Metabolism is essential for normal cellular function and dynamically changes to meet organism and tissue needs. Cardiac function and metabolism are closely intertwined. Fatty acid oxidation serves as the primary means to provide energy in normal conditions. In heart failure, cardiomyocytes lose this metabolic flexibility and become more reliant on glycolysis. Despite extensive work to understand the metabolic underpinnings of heart failure, more investigation is needed to dissect the underlying mechanisms of this disease. A class of metabolic diseases collectively known as inborn errors of metabolism (IEMs) provide a window into pathophysiology, including heart failure, due to their well-defined causes. Pathology arising from IEMs can be tracked back to a single mutation, providing a direct and tractable method of studying the disease. Lipoic acid deficiencies are a novel class of IEMs that cause metabolic decompensation, severe neurodevelopmental delays, and early death. Lipoyltransferase-1 (LIPT1) catalyzes the final step in de novo lipoic acid synthesis by transferring the lipoate moiety to 2-ketoacid dehydrogenases such as pyruvate dehydrogenase (PDH), oxoglutarate dehydrogenase (OGDH), and branched-chain ketoacid dehydrogenase (BCKDH). The Genetic and Metabolic Disease Program (GMDP) at UT southwestern has unique access to patient samples and clinical data. A LIPT1 deficient patient presented with neurodevelopmental delays and numerous unexpected metabolic phenotypes, including elevated serum 2-hydroxyglutarate (2HG), a metabolite with wide ranging impacts on cell signaling and epigenetic regulation. The patient also displayed altered cardiac function, including impaired systolic function and tachycardia secondary to atrial fibrillation, worsened by acute episodes of metabolic decompensation. We intend to characterize the underlying causes of cardiometabolic distress using novel mice to model cardiac LIPT1 deficiency. Mice lacking LIPT1 in the heart die within 6-7 weeks with severe systolic dysfunction and elevated levels of 2HG. The central hypothesis of this proposal is that LIPT1 deficiency pathologically limits cardiometabolic flexibility leading to deleterious metabolite accumulation, including 2HG, and impaired cardiac development and function. If successful, this proposal will generate a definitive assessment of cardiac LIPT1 deficiency in mice providing a detailed understanding of the metabolic consequences of this specific IEM. More broadly, this proposal will increase our understanding of the consequences of limited metabolic flexibility in cardiac tissue, a hallmark of heart failure. The appropriate usage of both patient data and mouse models will increase the disease relevance of the work discussed in this proposal.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The ability to remember one's own features and monitor the current states is a fundamental for self- recognition. To experimentally demonstrate the visual self-recognition, a mirror-induced visual self-recognition (MSR) test was developed for non-human primates, which requires animals to remember visual features of their own heads as a reference memory and recognize the current status via a mirror reflection. Human brain imaging and electrophysiological studies have suggested several brain regions potentially important for visual self-recognition. Especially, prefrontal cortex (PFC), posterior parietal cortex (PPC) and hippocampus (HPC) are currently considered to be crucial for visual self-recognition. However, how neurons, circuits and inter- regional network activity in the brain accomplish this remarkable function remains unknown, due to a limited availability of experimental animal models. Although the ability of MSR was initially reported in only few species, by modifying the experimental conditions to adjust to their nature, birds, fishes and rhesus macaques were also able to show MSR, suggesting that MSR may present in many more species than previously thought. Our goal is to examine the neural circuit mechanisms for MSR using a mouse model, focusing on how mice remember visual features of the self and recognize the current status via a mirror reflection. Our recent studies indicate that mice display mark-directed head-grooming to remove ink stains on their heads only when a mirror is visibly available. This mark-directed behavior requires long-term mirror habituation and social experience in the home cage. Our preliminary study with whole-brain neural activity mapping using immediate early gene expression revealed that both the medial PFC (mPFC) and HPC are activated during MSR in mice, suggesting that these brain regions involved in MSR are preserved across species. We found that chemogenetic inhibitions of ventral HPC impair MSR. Specifically, a subset of ventral hippocampal CA1 (vCA1) neurons is highly reactivated during exposure to a mirror, but not to other conspecifics, and is crucial for MSR in mice. We refer to these as self-responding neurons. Based on our preliminary data, previous human studies, and anatomical connections, our central hypothesis is that hippocampal-prefrontal cortical circuits may be crucial for MSR. In particular, we posit the conceptual framework that the visual self-image may be developed and stored in a subset of vCA1 neurons through social experiences and mirror habituation, while mPFC may facilitate visual self-monitoring for MSR by integrating the visual self-image from vCA1. To test these hypotheses, we will examine i) the roles of vHPC and mPFC neural activity on MSR (Aim 1), ii) the roles of self-responding vCA1 neurons on MSR (Aim 2) and iii) the roles of vCA1→mPFC pathway on MSR (Aim 3). We believe our proposed study is highly adventurous, because it will provide a first demonstration of the detailed functional map of the hippocampal-prefrontal cortical circuit that controls visual self-recognition.

NIH Research Projects · FY 2025 · 2024-09

Abstract Host defense responses by the mammalian immune system can be very potent but require exquisite spatio-temporal coordination. This coordination is essential to match the immune activity to the specific threat, to monitor and regulate the immune response, minimize damage to normal tissues, and terminate the response when the hazard is eliminated. Stimulator of interferon genes (STING) is an endoplasmic reticulum-associated signaling protein that is essential for transcriptional regulation of numerous host defense genes against malignant cells. STING is activated by 2’, 3’-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an endogenous secondary messenger, which is produced by cGAMP synthase (cGAS) in response to cytosolic DNA as a danger signal. Despite potent antitumor activities demonstrated by STING agonists in preclinical studies, early clinical trials have yet to show significant antitumor response in cancer patients. Current STING agonist designs are ‘always ON’, leading to on-target, off-tumor toxicity in healthy tissues. The proposed R35 program will integrate four areas of research to harness this important natural defense mechanism and innovate a safe and efficacious STING- targeted therapy for immune-resistant cancers. In nanotechnology, we will design and synthesize a (pH-hypoxia) AND logic nanoparticle STING agonist that stays protected in normal tissues but will be activated in response to acidic pH and hypoxia signals inherent in the tumor microenvironment. This ensures minimal toxicity in healthy tissues, while promoting targeted STING activation within malignancies. In STING signaling, we will employ cryo-electron microscopy to investigate the synergy of PSC7A with cGAMP for STING binding and activation. In dendritic cell biology, we will investigate STING-mediated transformation of hematopoietic progenitor cells into conventional type 1 dendritic cells (cDC1), and define its ramifications on antitumor immunity. In immune-oncology, we will employ patient-derived tumor fragments to probe into STING agonists' roles in immune resistant tumors. Furthermore, we will assess the prognostic value of the STING-cDC1 signature in forecasting therapeutic responses to treatments like STING-targeted interventions and checkpoint blockade therapies. Through our bench-to-clinic and back-to-bench approach, our goal is to pinpoint the barriers that have stymied effective targeting of this crucial biological pathway, and ultimately apply these insights to establish a successful STING-targeted therapy in cancer patients unresponsive to current treatments.

NIH Research Projects · FY 2025 · 2024-09

The problem of cancer in the 21st century remains a national priority, and as such, offers a substantive long-term career opportunity for pre-doctoral and post-doctoral training. The Translational Cancer Biology (TCB) T32 training program seeks support for 2 pre-doctoral and 4 post-doctoral trainees for 2 years each and emphasizes bench to bedside research encompassing state-of-the-art areas of cancer research. Along with training in the fundamentals of cancer research, trainees are provided with individualized access to many areas of basic and clinical cancer research along with training in the responsible conduct of research (RCR), including rigor in experimental design and data interpretation. Understanding how to work in multidisciplinary teams and being familiar with “systems biology” and large data set analyses are also a major goal of our training program. The TCB T32 program overarching goals are:  To train top-quality scientists capable of conducting independent cancer research.  To foster the intellectual, technical and communication skills required to succeed in the academic or industrial arenas of today and in the future.  To provide an understanding of the basic, public health, and clinical problems of cancer.  To provide a training program that includes cancer related didactic and journal-oriented courses as well as a program that includes biomedical ethics, RCR, enhanced reproducibility, biostatistics, bioinformatics and data mining, analyses and sharing.   To provide training in grant writing, effective oral communications, and career planning.  To maintain a program-specific recruitment program attracting a geographical broad range of applicants from a variety of scientific disciplines.   To have regular retreats to provide specific T32 opportunities for trainees to present their research and to develop the skills required to be effective scientists.  Our training grant eligible pre-doctoral and post-doctoral applicant pool has almost doubled during the last 5 years. The ability of this training grant to bridge an existing outstanding foundation of basic cancer research programs with our experimental therapeutics programs in the Harold Simmons NCI Designated Comprehensive Cancer Center, distinguishes it from a standardized general graduate and post-graduate educational program. We have 35 committed, well funded faculty mentors, with a dedicated group of steering committee members, along with Cancer Center administrative intellectual and financial support.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Preclinical models of lung cancer are essential tools for researchers to understand cancer biology and develop therapeutic strategies. Choosing the most cost-effective preclinical model to answer a specific scientific question requires careful study of the existing molecular and pathological characterization of different models and human tumors, as molecular profiling reveals the orchestration of biological processes and pathological characterization informs the spatial composition of the tumor microenvironment. While preclinical models of lung cancer have been extensively characterized, the molecular data remain scattered, the pathology data have rarely been deposited, and no tool exists to evaluate the molecular and pathological agreement between preclinical models and human tumors. We have previously built a lung cancer explorer, which provides user-friendly integrative analytical tools to explore gene expression and clinical data from over 6,700 patients in 56 published datasets. Leveraging patient lung tumor pathology image archives, we developed algorithms and pipelines to perform histopathology digital staining and feature extraction from H&E images and identified interesting pathology features that predict outcome and response to targeted therapy. Extending these efforts to preclinical models, this proposal aims to develop an informatics platform integrating molecular and pathology data from various lung cancer preclinical models and patient tumors to assess preclinical model fidelity through comparative analyses. Specific Aim 1 will harmonize molecular profiling datasets from various lung cancer preclinical models. Statistical methods for cross-study validation and quality control will be implemented to ensure computational compatibility and to select appropriate datasets for analysis. Model-specific web applications will be built to support data exploration and analysis. Specific Aim 2 will perform histopathological and spatial transcriptomic characterization of tumors from in vivo models. We will network with lung cancer preclinical model investigators to solicit contributions of pathology images and samples for establishing a public image archive and for spatial molecular profiling experiments. Effective algorithms and pipelines for preclinical model pathology image analyses will be established. Specific Aim 3 will integrate data collected and harmonized in Aims 1 and 2 to construct an informatics platform for cross-model comparison and alignment to human tumors. This platform will allow users to review and download our processed molecular and pathology datasets and compare molecular and pathology profiles of preclinical models and patient tumors from multiple facets. We will share these resources with the lung cancer research community and solicit feedback to improve our platform. The successful implementation of this project will assemble the scattered molecular datasets, establish a large- scale public pathology image and spatial molecular profiling resource, and establish a user-friendly integrative fidelity assessment platform for lung cancer preclinical models.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The goal of this K08 application is to provide a rigorous 5-year scientific and career development training plan that will facilitate the transition of Dr. Glynnis Garry from a post-doctoral fellow to a fully independent investigator as a cardiovascular physician-scientist. Dr. Garry obtained her medical degree from Vanderbilt University during which she pursued a rigorous and productive 1.5 year molecular biology training through a Sarnoff Cardiovascular Research Fellowship at UT Southwestern in the laboratory of Eric Olson, Ph.D, one of the world’s leading experts in muscle biology and cardiovascular regeneration. After completing a short-tracked Internal Medicine residency at UT Southwestern through the Physician-Scientist Training Program, Dr. Garry joined the laboratory of Dr. Olson as a post-doctoral fellow, where she directed her focus to investigating direct cardiac reprogramming as a means of promoting heart regeneration following myocardial infarction. Reprogramming of fibroblasts to cardiomyocytes has emerged as an attractive strategy to redirect the fibrotic response of the injured, non-regenerative heart toward a functional myocardium. However, previously discovered cocktails have been ineffective in reprogramming adult human fibroblasts, limiting the clinical application of this strategy. From a large unbiased screen, Dr. Garry identified the histone reader PHF7 as the most potent activator of adult reprogramming. Her preliminary work demonstrated the ability of PHF7 to markedly activate reprogramming in adult human and mouse fibroblasts by increasing chromatin accessibility at cardiac super enhancers when added to a five-factor reprogramming cocktail. She also discovered the ability of PHF7 to activate adult murine reprogramming in the absence of canonical reprogramming factors. Further, she recently generated data demonstrating the ability of PHF7 to improve cardiac function following myocardial infarction, which is an unprecedented result that merits further characterization. In her proposed research, Dr. Garry under the mentorship of Dr. Olson will aim to 1) define the role of PHF7 in adult human reprogramming, 2) define the ability of PHF7 and PHF7 cocktails to improve cardiac function following myocardial infarction, and 3) delineate mechanisms by which PHF7 induces reprogramming events and improvement in cardiac function in vivo. These studies will potentially provide the basis for development of a novel therapeutic factor for the treatment of ischemic heart disease and advance our understanding of mechanisms that drive reprogramming. Dr. Olson is a pioneer in the field of cardiac reprogramming with a storied legacy of mentorship. He is fully committed to mentoring the career of Dr. Garry, and together they have assembled an outstanding advisory committee to ensure her success through scientific mentorship, grantsmanship, coursework, and career development opportunities. Given her outstanding mentorship, productive research program, and full institutional support and resources of UT Southwestern, Dr. Garry is well-positioned to launch a successful career as an independent leading academic cardiovascular physician-scientist and submits a K08 award application to this end.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Mitochondria are essential organelles that serve as the cellular hub for metabolism, ATP production, and redox signaling. We identified a novel mechanism of signaling in the mitochondria mediated by a post translational modification known as AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates. Our previous studies revealed that Selenoprotein O catalyzes AMPylation of multiple mitochondrial proteins involved in redox homeostasis and cellular metabolism. There are several critical gaps in our current knowledge of AMPylation including the functional importance of AMPylation, as well as the enzymes that reverse AMPylation. To gain a mechanistic understanding of AMPylation, we developed an enrichment strategy for AMPylated proteins and identified RNase Z as a deAMPylase that catalyzes the removal of AMP from AMPylated substrates. RNase Z was previously shown to be a conserved endoribonuclease that cleaves the 3’ trailer of precursor tRNAs to generate mature tRNAs. Our studies establish RNase Z as a multifunctional protein with previously unrecognized functional roles beyond tRNA processing. Thus, the goal of this proposal is to determine the biochemical and molecular mechanisms of RNase Z-mediated deAMPylation. Mutations in RNase Z result in severe hypertrophic cardiomyopathy and increased prostate cancer susceptibility. We hypothesize that the deAMPylation activity, in addition to the endoribonuclease activity, contributes to the functional importance of RNase Z in the mitochondria. However, our understanding of mitochondrial AMPylation is in its infancy due to the lack of tools to study the AMPylated proteins. Thus, we developed a novel enrichment strategy for the identification and functional characterization of AMPylated proteins. We anticipate these studies will reveal previously undocumented roles for AMPylation in cellular signaling and the molecular mechanisms of RNase Z-associated diseases.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Protein sequences can be broadly categorized into two classes: those which adopt stable secondary structure and fold into a domain (i.e., globular proteins), and those that do not. This latter class of sequences are conformationally heterogeneous and are described as being intrinsically disordered. Structural biology has enabled the development of bioinformatic approaches that can sub-classify globular sequences by domain type, an approach that has revolutionized how we understand and predict protein functionality. Conversely, it is unknown if protein intrinsically disordered regions (IDRs), which cannot be resolved by structural biology, are subject to broadly generalizable organizational principles that would enable their sub-classification and the a priori prediction of function. Protein Low Complexity Domains (LCDs) are a class of IDRs enriched in a small subset of amino acids. By simply gazing at LCDs, local sequence biases are evident. We hypothesized that the non-uniform distribution of amino acids with LCDs may be a conspicuous manifestation of a more general organizational principle widely operative in IDRs. We therefore developed a statistical approach that quantifies linear variance in amino acid composition across a sequence. This algorithm has led to the surprisingly discovery that IDRs are non-randomly organized into juxtaposed modules of distinct compositional bias. This type of sequence organization is present across the three domains of life and in IDRs of both low and high sequence complexity. Our data show that this sequence organizational principle is broadly operative and suggest a hitherto unappreciated level of logic and interpretability in this enigmatic class of sequences. Motivated by these observations, this proposal seeks to use the logic of modularity to comprehensively classify IDRs, develop a predictive understanding of IDR function, and define how the modular architecture of disordered sequences impacts their conformation and function. In Aim 1 we will use computation and quantitative metrics to categorically cluster modules in order to assess both the evolutionary diversification of IDRs and to relate module types (and thus IDRs) with specific functions. In Aim 2 and Aim 3 we will undertake in vitro and in vivo functional studies targeting two model disordered sequences. Specifically, we will determine how the unique modular structure of a DNA-binding IDR and a desiccation-tolerant IDR relates to their in vitro conformation and function using biochemical assays and related in cellula approaches. Altogether, these studies will provide the first comprehensive classification of IDRs to enable a priori functional predictions for disordered sequences, and the proposed functional studies will delineate how modularity impacts conformation and function. Altogether, this work will usher in a new level of interpretability and predictability in our understanding of the functional mechanism of IDRs.

NIH Research Projects · FY 2025 · 2024-09

Project Summary This proposal presents a five-year research career development award for Dr. Anna Rivara, an Associate Research Scientist (Yale's equivalent to Assistant Professor (Research)) in the Department of Chronic Disease Epidemiology at the Yale School of Public Health (YSPH). The proposal aims to address important issues surrounding the current epidemic of Type 2 Diabetes Mellitus (T2D) being experienced by Samoans and other Pacific Islanders. Project goals are to 1) examine preferences for diabetes care among adult Samoans recently diagnosed with diabetes using a discrete choice experiment, 2) prospectively estimate the associations between diabetes care and glycemic outcomes among adult Samoans, and 3) longitudinally assess diabetes care decision-making and coping strategies. Outcomes from this project will be integral to understanding and offering effective and acceptable treatment options to alleviate the burden of diabetes faced by Pacific Islander populations. The proposal describes a rigorous, mixed methods-based investigation that will be made possible through the advanced mentoring and training that the PI will receive throughout the award period. The proposed work will be conducted under primary mentors Dr. Nicola Hawley, Ph.D, Associate Professor of Epidemiology (Chronic Diseases) at YSPH, Dr. Leslie Curry, Ph.D., MPH, Professor of Health Policy at YSPH and Associate Director of the Yale Scholars in Implementation Science Training Program, and Dr. Satupaitea Viali, MBBS, MPH, of the National University of Samoa and the University of Otago. Additionally, collaborators Dr. Omar Galárraga, Ph.D, Associate Professor of Health Services, Policy and Practice at Brown University School of Public Health, Dr. Rochelle Rosen, Ph.D., Associate Professor of Behavioral and Social Sciences at The Miriam Hospital and Brown University, Dr. Erin Kershaw, Ph.D., MD., Associate Professor of Medicine at the University of Pittsburgh, and Dr. Jenna Carlson, Assistant Professor of Biostatistics and Human Genetics at the University of Pittsburgh, will be providing mentorship and hands-on training in research implementation, dissemination, and mixed methods analyses. Dr. Rivara's training goals are to 1) gain new skills in discrete choice experiments and longitudinal analytical approaches and advance skills in mixed methods data collection and mixed effects linear modeling; 2) strengthen professional development through preparation and submission of policy briefs and peer-reviewed manuscripts and science communication via presentation of results at invited seminars, and national and international conferences; and 3) advance skills in grantsmanship though didactic and applied training and the construction and submission of a diabetes-focused R01 intervention upon the completion of the award period. Together, the research outcomes and training will lay the foundations for Dr. Rivara's career developing culturally sensitive, participant-guided interventions in low resource settings as a diabetes-focused health services researcher.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Pineoblastoma is one of a family of childhood cancers that can arise through mutations in microRNA processing genes DROSHA and DICER1. It is unknown whether these tumors develop through shared or distinct oncogenic pathways, and there are no ways to therapeutically target such mutations. To understand how these mutations cause cancer, we developed a mouse model of pineoblastoma by ablating Drosha or Dicer1 in the developing pineal gland. These mice develop tumors that resemble human pineoblastoma. They are highly proliferative; they exhibit an expression signature of the embryonic pineal gland; and they overexpress microRNA target genes. Among the microRNA target genes overexpressed in these tumors is a set of developmental transcription factors, including Onecut2. Furthermore, the binding sequences recognized by these transcription factors is enriched in regions of open chromatin in these pineal tumors. In this project, we will investigate the role of Onecut2 in the developing pineal gland and in pineal tumor development. In Aim 1, we test whether Onecut2 is necessary for tumor development. In Aim 2, we investigate how Onecut2 interacts with other neurodevelopmental transcription factors. In Aim 3, we explore whether ONECUT2 is required in patient-derived models of pineoblastoma. If successful, this project would demonstrate that mutations in DROSHA or DICER1 can arrest differentiation through developmental transcription factors and that impairing the activity of these transcription factors could be a future therapeutic strategy.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY There is growing evidence that some diseases can be influenced by biological sex, where male or female patients have worsened outcomes or faster disease progression. However, the molecular mechanisms underlying the interaction of systemic sex hormones and disease are largely unknown. There is a critical need to understand the molecular basis of how circulating sex hormones can impact disease progression. I have shown that a mouse model of rhodopsin P23H Retinitis Pigmentosa (RP) presents as sexually dimorphic, where disease progression is increased in females compared to males. RP is a group of devastating visual disorders that cause the death of the photoreceptor neurons, leading to blindness. In addition to my findings of sexual dimorphism in retinal disease progression in RP, I have discovered that this sex difference can be ameliorated by depletion of estrogen and progesterone. Re-introduction of either of these sex hormones can worsen visual decline. The overall goal of my work is to understand the mechanisms that influence photoreceptor cell death in RP. Specifically, this proposal aims to determine how systemic sex hormones impact cell death and stress responses in the rhodopsin (Rho) P23H form of RP. To do so, I will first employ targeted molecular biology techniques to analyze the effect of circulating estrogen and progesterone on the unfolded protein response and caspase- mediated cell death. By investigating these two pathways, I will describe how the hormones that are detrimental to photoreceptor health in RP are affecting two of the central molecular mechanisms underlying this disease. Next, I will investigate how these hormones are able to carry out their detrimental functions by defining the hormone receptors that mediate this response through in vivo antagonist/ agonist studies. Furthermore, I will determine the genetic control of the hormone signaling in the retina through RNA sequencing. Together, these aims will inform how and what is being altered in the Rho P23H retina in response to hormone signaling. Successful completion of this project will be vital for informing the clinical safety of hormonal medications for patients with this form of RP, as well as discovering new pathways that can affect photoreceptor degeneration. Furthermore, this work provides me the opportunity to advance my skills in the study of hormone signaling and cellular stress and promotes my career goals of becoming an independent investigator.

NIH Research Projects · FY 2025 · 2024-09

PI: Mitra, Sharmistha Glycogen is the largest soluble macromolecule in the cell and serves as a critical energy store in tissues. Co- ordinated actions by multiple enzymes control regular branching, radial spherical growth of the molecule ensuring the solubility, which is critical for tissue homeostasis. Regulatory enzymes of the glycogenesis process includes at least three E3 ubiquitin ligases, dedicated to securing glycogen’s spherical architecture. Absence of any of them leads to a glycogen structure akin to the insoluble starch amylopectin (now called polyglucosans). This process of forming polyglucosans is called amylopectinosis. Polyglucosans tend to aggregate producing insoluble deposits called polyglucosan bodies (PB). At the tissue level, precipitated PBs lead to untreatable pathologies such as fatal cardiomyopathy (in heart), disabling skeletal-myopathy (muscle), motor neuron loss (central nervous system) and neurodegeneration (brain). How deficiencies of each of these ubiquitin ligases (or their interacting proteins) and through what substrates and pathways, result in the derangement of glycogen structure is not known. In the current proposal, the PI proposes to work with Linear Ubiquitin Chain Assembly Complex (LUBAC) to identify its role in glycogen solubility control. LUBAC is a multi- protein complex composed of two E3 ubiquitin ligases – RBCK1, HOIP and an adaptor protein SHARPIN. LUBAC-deficient patients exhibit PBs in different organs, especially skeletal and cardiac muscle resulting in myopathy and cardiomyopathy with heart failure. This emphasizes LUBAC’s important role in glycogen metabolism. To date, glycogen metabolism related LUBAC substrate(s) and associated molecular mechanisms are not known. Utilizing newly created mouse models, cell lines and novel approaches, the proposed work tests the central hypothesis that LUBAC downregulates the activity of glycogen synthase (GS) and ubiquitinates longer less-branched glycogen chains to keep the glycogen soluble. Aim 1 of the research uses two newly created mouse models of LUBAC deficiency to understand Rbck1’s glycogen association. Furthermore, to understand the mechanisms of amylopectinosis, the PI investigates the influence of glycogen phosphate, and muscle/brain cell specificity for PB accumulation. In Aim 2, utilizing newly created overexpression cell lines and mouse models, a detailed mechanistic pathway by which Rbck1 deficiency results in high GS activity is tested. Additionally, aim 2 by using a novel in-vivo approach, tests the possibility of Rbck1 directly ubiquitinating long, precipitation-prone glycogen chains. Finally, in aim 3, using novel cell models and quantitative proteomics, the proposal identifies additional pathways/substrates for GS activation. Taken together, the proposed aims comprehensively study LUBAC’s function in glycogen solubility with novel understandings of its role in amylopectinosis. Deeper understandings of LUBAC mediated control of glycogen metabolism will not only provide new insights towards developing a treatment for a fatal rare condition, but also have implications in other common disease research such as cancers and Alzheimer’s disease where both glycogen metabolism and LUBAC are often dysregulated.

NIH Research Projects · FY 2025 · 2024-09

Project summary/abstract Autism spectrum disorders (ASD) are estimated to affect 1 in 36 children in the US, yet treatments for ASD remain limited. Mutations in the transcription factor, FOXP1, have been linked to ASD, yet the precise role for Foxp1 in either neurodevelopment or ASD remains incomplete. Previous studies of ASD have also shown that the cortex may exhibit disrupted structure and function. In line with this, we previously created a forebrain specific Foxp1 knockout mouse model and observed that there were behavioral abnormalities including social deficits, communication impairments, and hyperactivity. However, the molecular mechanisms that result from Foxp1 loss in early neocortical development remain unclear. Given the cell-type specific expression pattern of Foxp1 during cortical development, understanding the cell specific function of Foxp1 is critical for the advancement of our understanding of neurodevelopmental disorders. Here, I propose to evaluate the impact of Foxp1 loss on two aspects of early neocortical development: migration and cell fate. First, I hypothesize that Foxp1 loss disrupts radial migration of upper layer neuronal subtypes through their interaction with aRGC fibers. I will test this hypothesis by utilizing 1) BrdU birth dating to track migration of neurons from the VZ along with 2) GFAP staining of aRGC fibers and 3) MERSCOPE’s capacity for detecting up to 1000 cell-type markers at a single time to determine the cell-type specific impact of Foxp1 loss on neuronal migration during upper and lower layer cortical neurogenesis. Concurrently with this, I will also stain for genes identified as downstream targets of Foxp1 through the collection of snRNAseq and CUT&RUN data in order to determine the impact of Foxp1’s downstream targets on migration. Secondly, I hypothesize that Foxp1 loss disrupts the timeline of neurogenesis causing changes in neuronal cell fate. In Aim 2, I will test this hypothesis by leveraging data from Aim 1 to assess the impact of Foxp1 on cell-type and cell fate during canonical upper layer and lower layer neurogenesis. I will do this by comparing the birthtime of a neuron, indicated by BrdU, to its cell-fate after two or after seven days. I will also repeat experiments from Aim 1 during the proliferative and gliogenesis phases of cortical development to determine whether cell fate is altered. By completing these aims and receiving training in the methods needed to generate and analyze these data, I will determine which cell types are most vulnerable to migration and cell fate deficits in a model system relevant to ASD. I will also provide key insights into the mechanisms of neurodevelopment, migration, and cell fate in the developing embryonic cortex. This will ultimately provide a critical foundation for the advancement of precise cell type targeted genetic therapies of neurodevelopmental disorders. Furthermore, I will receive training in cutting edge wet bench technologies, including cellular resolution spatial transcriptomics, CUT&RUN, and snRNAseq while gaining tremendous experience as a bioinformatician. This expertise will enable me to continue to advance our understanding of cell type-specific genetic contributions to development and disease throughout my career.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY (ABSTRACT) Early-onset colorectal cancer (EOCRC; CRC diagnosed at < 50 years of age) is a known and worsening public health problem, with alarming increases in incidence across the US, further complicated by lack of healthcare access and poor outcomes among racial and ethnic minorities. Diagnosis for younger patients must rely on symptomatic presentation or another trigger, as routine screening is not recommended for them. Asymptomatic triggers for CRC testing are family history of CRC, precancerous polyps, genetic predisposing syndrome, inflammatory bowel disease, but these represent a small proportion of cases. A much larger proportion of cases have patient report of “red-flag” symptoms (rectal bleeding, abdominal pain, unintentional weight loss, constipation/diarrhea, or iron-deficiency anemia). However, even in the presence of these symptoms, factors at multiple levels contribute to delayed diagnosis: low physician suspicion of cancer, failure to recognize concerning symptoms, overlap of symptoms with other benign disorders, and patients’ lack of knowledge about the disease. Consequently, younger patients are more likely to be misdiagnosed, experience diagnostic delays, report longer duration of symptoms prior to testing, and be diagnosed at later stages. To facilitate earlier diagnoses of EOCRC, we need to better understand prevalence of CRC test orders and receipt for younger symptomatic patients, and explore the association of time-to-testing with cancer diagnoses, especially among uninsured, underserved, minority patients. We have a unique opportunity to generate important information about CRC test order and testing in light of red-flag symptoms among a large and vulnerable group of younger safety-net patients seen in Dallas County’s Parkland Health system. We will use existing electronic health records data (EHR) for patients aged 40-49 years from the current NIH-funded PROSPR cohort study, and extend it by extracting additional Parkland EHR data for those aged 18-39 years, to develop a curated comprehensive colorectal timeline file for those in the “early onset” age range of 18-49. Using data from these under- and uninsured minority patients, we aim to describe EOCRC symptom burden and healthcare system interactions leading to CRC test order and test receipt (Aim 1), and explore association of time-to-testing with cancer stage among patients with EOCRC (Aim 2). Results from this study will generate epidemiologic evidence about CRC testing among younger under- and uninsured symptomatic patients, with efficiency gained from use of 10 years of data from a current NIH- funded cohort study and resultant algorithms. Results from this work can guide development of recommendations generalizable to a diverse group of people, and generate future research questions to understand appropriate EOCRC screening and follow-up in this vulnerable population.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Syphilis, caused by Treponema pallidum spp pallidum (T. pallidum), cases are rising at an alarming rate with devastating consequences for pregnant individuals and neonates. Syphilis in pregnancy carries a 21% increased risk for stillbirth, 6% increased risk for preterm delivery, and 9% increased risk for neonatal death. Some neonates with congenital syphilis manifest symptoms of disseminated infection before 2 years of age (early congenital syphilis) which carries a mortality rate of up to 38%. However, most neonates (60-90%) are asymptomatic at birth, and may develop symptoms of congenital syphilis later in life with risks for permanent damage to nervous, bone, eye, and musculoskeletal systems. There is currently no molecular diagnostic test for congenital syphilis in routine clinical use. Instead, indirect diagnosis of possible or probable infection relies heavily on neonatal serologic titer relative to maternal titer and maternal treatment history. Due to risks associated with a missed diagnosis of congenital syphilis and the lack of accurate diagnostics, treatment decisions are frequently subjective. There is a critical need for accurate, sensitive, and direct detection of syphilis infection to better inform diagnosis and treatment for congenital syphilis. Polymerase chain reaction (PCR)-based molecular tests have become routine for the detection of numerous infectious diseases due to the sensitivity and specificity of the technique. Sensitivity of PCR differs according to gene target, specimen type, and sampling technique. Despite attempts to develop PCR targeting regions of conserved genes like PolA and Tp47, no FDA approved syphilis PCR test exists. There is a gap in our understanding of the optimal sampling strategy for PCR as an adjunct to current algorithms for diagnosis of congenital syphilis and risk stratification, particularly in asymptomatic neonates. Therefore, we hypothesize that molecular PCR performance can be optimized by determining the best diagnostic specimen(s) for congenital syphilis in the context of maternal stage, treatment history and neonatal clinical evaluation. We have assembled a team to optimally address this hypothesis. Dr. Emily Adhikari is a maternal-fetal medicine specialist and Medical Director of Perinatal Infectious Diseases at Parkland Hospital where she supervises diagnosis and treatment of syphilis in pregnancy. In collaboration with neonatologist Dr. Shamaila Gill, and pediatric infectious disease specialist Dr. Amanda Evans, comprehensive neonatal diagnostic evaluation and clinical staging will be performed. Dr. Jeff SoRelle is trained in molecular genetic pathology and the requirements to clinically validate a test. He has developed several molecular tests including COVID-19 variants, which led him to collaborative efforts of the impact of Delta and Omicron variant infections in pregnancy with Dr. Adhikari resulting in publications in JAMA and Am J Obstet Gynecol. We propose the following aims: AIM 1: Determine whether PCR detects T. pallidum DNA in neonates exposed to maternal syphilis. AIM 2: Determine the best sample type and source for T. pallidum PCR.

NIH Research Projects · FY 2024 · 2024-08

Glucagon-like peptide 1 (GLP-1)-based therapeutics have profound effects on body weight and blood glucose management. GLP-1 cells are located in both the periphery and the caudal medulla, specifically within the nucleus tractus solitarius (NTS). However, the effects of GLP-1 or long-acting GLP-1 receptor agonists (GLP1Rags) on synaptic/cellular properties in the brain and their contribution to metabolic changes are not entirely understood. Based on pilot data, we hypothesize that a DMH GLP-1R → NPY/AgRP neuron circuit is a target for brain-derived GLP-1 neurons and GLP-1Rags. Our objective is to determine if DMH GLP-1R neurons are required for the NTS GLP-1-induced effects on NPY/AgRP activity and metabolism. The project will use chemogenetics, electrophysiology, and in-vivo calcium imaging to investigate these questions. These experiments will potentially bridge our understanding of the regulation and physiological roles of the GLP-1system in the brain and in the treatment of metabolic disease. Glucagon-like-peptide-1 receptor agonists (GLP-1Rags) have profound anti-diabetic and antiobesity effects, but the neural systems responsible for mediating these effects are not fully understood. The endogenous function of brain-derived GLP-1, located in the Nucleus Tractus Solitarius (NTS), may provide insight into the effects of GLP-1Rags in the brain. In particular, NTS GLP-1 neurons target multiple brain regions and reduce food intake and glucose production upon activation, which is similar to the effects of GLP-1Rags. While NTS GLP-1 neurons do not express GLP-1receptors, GLP-1Rags may activate target nuclei downstream of NTS GLP-1 neurons that do express GLP-1Rs, thereby mimicking the effects of stimulating NTS GLP-1 neurons. We hypothesize one component of the beneficial effects of both NTS GLP-1 and GLP-1Rags on metabolism involves an NTS GLP-1 neuron → dorsal medial nucleus of the hypothalamus (DMH) GABAergic neuron → Arcuate nucleus circuit that ultimately reduces Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neuron activity. This study aims to define this NTS → DMH → arcuate circuit and determine if GLP-1Rags and central GLP-1 converge on the DMH to inhibit NPY/AgRP neurons and improve metabolism. Previous work and preliminary data demonstrate that: 1) GLP1-Rags decrease the excitability of NPY/AgRP neurons, 2) a GABAergic neuron that expresses GLP-1Rs and leptin receptors (LepRs) resides in the DMH, 3) DMH GLP-1R+ and LepR+ neurons are activated by GLP-1Rags, food presentation/intake, and/or elevated glucose levels, and 4) there is synchrony in the regulation of activity of these neurons. Specifically, when NTS GLP-1 or DMH LepR+/GLP-1R+ neurons are activated, NPY/AgRP neurons are inhibited. These findings suggest the NTS GLP-1 → DMH GLP-1R/LepR → NPY/AgRP circuit functions as a convergence point for satiety signals. We aim to investigate the role of DMH GLP-1R+ neurons in mediating the effects of GLP-1Rags and NTS GLP-1 neuron activity, as well as explore how this circuit affects NPY/AgRP neuron activity and metabolism. The proposed model (Figure 1) predicts that activation of DMH GLP-1R neurons by GLP-1Rags or by NTS GLP-1 neuronal projections (e.g. in response to a meal or in response to exercise) leads to increased GABA release from DMH GLP-1R neurons and inhibition of NPY/AgRP activity. This, in turn, mediates various beneficial metabolic effects of central GLP-1 and GLP-1/Rags. The studies outlined below are designed to directly test the predictions. Specific Aim – To determine if increased activity of NTS GLP-1 neurons alters DMH and arcuate NPY/AgRP neuron physiology and metabolism. We hypothesize that NTS GLP-1 neuron activation leads to inhibition of arcuate NPY/AgRP neurons through a GABAergic DMH interneuron that expresses GLP-1Rs and/or LepRs. We will utilize optogenetic and chemogenetic approaches to activate NTS GLP-1 neurons while simultaneously monitoring DMH GLP-1R+ or LepR+ neurons as well as arcuate NPY/AgRP neuron activity exvivo (electrophysiology) and in-vivo (calcium imaging). Summary: The proposed studies link the GLP-1R agonist class of anti-obesity/anti-diabetes medications to a novel NTS GLP-1 → DMH GLP-1R/LepR → NPY/AgRP circuit. We will examine the requirement of GLP-1 neurotransmission and GLP-1R signaling in the plasticity of this circuit using patch clamp electrophysiology, invivo calcium imaging, and metabolic phenotyping with unique mouse models already in hand