Baylor College Of Medicine

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT The escalating use of kratom, an herbal substance with opioid-like properties, has raised significant public health concerns due to its potential for abuse and dependence. Despite a drastic increase in calls to poison control centers regarding kratom-related incidents, human laboratory studies investigating its abuse potential are lacking. The overall objective of this application is to address this gap by conducting a comprehensive evaluation of the abuse potential and pharmacokinetic (PK) profile of kratom in humans. The study will employ a rigorous, randomized, double-blind, crossover design involving non-treatment seeking recreational opioid users (n=60). Participants will complete 1 of 6 pre-specified treatment sequences according to a balanced 6 x 6 Latin square design; each dosing sequence consists of 6 dosing periods that evaluate the abuse potential of 1 of the 6 study treatments, including kratom at different doses, positive control drugs, and placebo. The primary objective is to determine whether kratom elicits subjective effects distinct from those produced by the controls, utilizing standardized visual analog scales (VAS) to assess “drug liking” and other subjective measures. Additionally, the project seeks to characterize the PK profile of kratom following oral administration, focusing on parameters such as absorption, distribution, metabolism, and elimination kinetics. Blood samples will be collected at specified intervals to analyze kratom and its major metabolites, correlating these findings with subjective and physiological effects over time. The anticipated outcomes of this research include valuable insights into the abuse potential and risks associated with kratom use, informing regulatory decisions regarding its scheduling and labeling. By providing comprehensive PK data on kratom, this research will contribute to a better understanding of its pharmacological profile and aid in safeguarding public health against the potential harms of kratom misuse and abuse.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Obesity cultivates complex immune responses in white adipose tissue (WAT), which may contribute to the development of insulin resistance and type 2 diabetes mellitus (T2DM). However, there still exists a gap in knowledge on cellular mechanisms regulating “obesity-specific” immune responses that precludes the development of novel immune therapies to treat obesity and related insulin resistance and T2DM. We were one of the first to report T cells in WAT in obesity. Further studies from us and others showed that WAT conventional T cells promote, and regulatory T cells (Tregs) protect against, insulin resistance in obesity. Nonetheless, the mechanisms underlying WAT Treg homeostasis and mediating their metabolic benefits remain incompletely understood. This application aims to investigate microRNA-30a (miR-30a) regulation of WAT Tregs and its impacts on WAT immune response and metabolism in obesity. This proposal builds on our novel pilot data: 1) miR-30a expression in WAT correlated positively with insulin sensitivity in obese mice and people; 2) enforced miR-30a expression in WAT promoted whole-body insulin sensitivity in obese mice; 3) miR-30a levels were high in WAT Tregs in lean but reduced in obese mice; 4) enforced miR-30a expression in WAT of obese mice increased WAT Treg numbers; 5) specific overexpression of miR-30a in Tregs increased WAT Treg numbers and improved insulin sensitivity in mice fed high-fat diet. Therefore, we hypothesize that miR-30a in WAT stimulates expansion of WAT Tregs and improves Treg functions to increase insulin sensitivity. We will test this hypothesis by pursuing three aims: Aim 1 will define effects of cell (Treg vs adipocyte)-restricted miR- 30a on WAT immune response and metabolism in obesity and establish cell-specific roles of miR-30a; Aim 2 will build on our additional pilot data that miR-30a delivery in WAT suppresses STAT1 (a key inflammatory transcription factor) and establish how miR-30a targeting of Stat1 governs WAT Treg homeostasis in obesity; Aim 3 will build on our novel data that compared to diet high in saturated fat, diet high in unsaturated fat induces higher levels of WAT miR-30a and Tregs in mice and examine dietary regulation of miR-30a and its role in WAT immune response and metabolism. We will accomplish these aims using gain-of-function and loss-of-function approaches in genetically modified animal models, cutting-edge immunophenotyping and transcriptomics, gold standard clamp for insulin sensitivity, and other state-of-the-art tools. Upon completing these aims, we expect to have uncovered a novel mechanism that may help develop new therapies that leverage the immune system to treat obesity and T2DM.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The pathogenesis of Parkinson’s disease (PD) and related synucleinopathies such as dementia with Lewy bodies are characterized by progressive deposition Lewy bodies and Lewy neurites composed primarily of phosphorylated alpha- synuclein aggregates (α-syn), neuroinflammation, blood-brain barrier compromise, and neurodegeneration. Currently, there are no clinically approved biomarker diagnostic tools or disease modifying treatments for PD. Clinical diagnosis is based on a combination of motor and nonmotor symptoms, but the neurodegeneration process may precede the first appearance of these symptoms by decades. Empirical data suggests that levels of oligomeric α-syn in the cerebrospinal fluid (CSF) of PD patients surpass those of normal individuals by several orders of magnitude. Oligomeric α-syn and reactive gliosis are strongly implicated in the initiation and spread of the disease. Recent data from clinical trials with anti-amyloid-β (anti-Aβ) monoclonal antibodies (mAbs) in Alzheimer’s disease (AD), (despite lack of clarity in the full safety/efficacy profile), suggest that anti-Aβ mAbs statistically improved cognitive and biomarker outcomes, demonstrating that neurodegenerative amyloidosis disorders can be slowed or halted. Over the past decade, we have demonstrated in several mouse models of neurodegeneration that following tail vein injection, liposomes bearing an imaging contrast payload cross the BBB into the CSF, primarily at the blood-CSF barrier at the at the choroid plexus and cerebrovascular leaks and bind to specific targets within the brain, enabling separation of disease mice from controls using noninvasive magnetic resonance imaging (MRI) or computed tomography imaging. Our lead product is currently in clinical trials as the first MRI-based imaging agents for Aβ plaques in AD. In a recent proposal funded by the NIA (R21 AG067131), we hypothesized that a variant of our agent labeled with oligomeric α-syn ligands will act as a scavenger for the pathologic species resulting in the formation of cross- linked agglomerates of the agent and pathologic species making them more suitable substrates for rapid phagocytosis (sizes > 0.5 microns) by activated glia. This in turn can lead to momentary accumulation of detectable levels of the agent in PD positive brains enabling noninvasive separation of test subjects from controls. Our preliminary data demonstrates successful in vitro formation of agglomerates upon exposure of the agent to α-syn fibrils and accelerated uptake of the ensuing agglomerates by microglia and neuroblastoma cell lines. In vivo administration by tail vein injection followed by MRI showed statistically significant signal enhancement in the brains of transgenic mice versus controls. The in vivo data was verified by ex-vivo immunohistochemical analysis which showed a correlation between in vivo MRI signal, regional distribution of the agent in brain tissue, Lewy pathology, and microglia activity. In this application, we seek to capitalize on this data and our experience in the area to fully establish the preclinical potential of our nano scavenger concept both as diagnostic and therapeutic strategies for PD. Specifically, we propose to test the efficacy of the agent to profile/halt disease progression from the prodromal stage through early to late stages in the M83 α-syn transgenic mouse line. The in vivo data will be validated with ex vivo histology analysis and behavioral studies. Furthermore, we propose to establish more insights on the cellular mechanisms involved in uptake and degradation of the biomarker using both murine and human cell lines.

NIH Research Projects · FY 2025 · 2024-09

Project Abstract/Summary Sudden infant death syndrome (SIDS) is an idiopathic disease characterized by an unknown cause of death in infants less than a year of age after a full autopsy, death scene investigation, and medical history review. SIDS is a large subset of Sudden Unexpected Infant Death (SUID) which, according to the CDC, takes the lives of 3400 infants per year in the US alone and is one of the leading causes of deaths in neonates representing a significant, unmet medical need. Although the underlying cause of SIDS remains unknown, carefully controlled post-mortem studies have revealed subtle brainstem abnormalities to be present in many SIDS cases. Researchers have found that, in subsets of SIDS cases, there are significant decreases in serotonin (5- Hydroxytryptomine, 5-HT), tryptophan hydroxylase 2 (TPH2) levels (an enzyme necessary for 5-HT production), and decreased binding affinity of some 5-hydroxytryptamine receptors. Notably, in some of the same SIDS cases showing reduced serotonin levels, researchers also found a significant increase in the number of serotonergic neurons (5-HT neurons), suggesting a negative feedback relationship between developmental serotonin signaling and neurogenesis to regulate serotonergic neuron numbers. However, the functional consequences of these serotonergic abnormalities associated with SIDS remains to be fully characterized. A leading hypothesis in the field is that the failure in the autoresuscitation reflex is a common endpoint in many SIDS cases. Autoresuscitation occurs when the infant stops breathing and falls into an apneic state due to extreme hypoxic/hypercapnic conditions, typically thought to occur from lying in a face down position, as many SIDS cases are found. As a last line of defense, the infant initiates deep gasping to reoxygenate the heart to restart its cardiorespiratory system. Studies have shown that the loss, or acute perturbation, of 5-HT signaling and/or neurons in neonate mice significantly impacts their ability to autoresuscitate or survive when they are exposed to episodic anoxia, mimicking SIDS like, face down conditions. However, it is not clear which serotonin receptors (5-HTr) play a role in the autoresuscitation reflex. I hypothesize that the loss of Htr1B function negatively impacts the neonate autoresuscitation reflex and embryonic serotonergic neurogenesis, two key processes thought to be functionally disrupted across a large subset of SIDS cases. To test my hypothesis, we will utilize loss of function and conditional Htr1B knockout mouse lines in combination with our closed loop automated robotic platform to characterize neonate cardio-respiratory function and autoresuscitation reflex. Additionally, we will assay mutant mice using novel spatial transcriptomics (MERFISH) to examine activity dependent genes as a proxy for respiratory network dynamics as well as genetic lineage tracing tools to characterize developmental changes related to serotonergic neurogenesis. Successful outcomes of the work will mechanistically link Htr1B to the neonate autoresuscitation reflex and 5-HT neuron development yielding critical insights about SIDS and clearing a path toward potential diagnostic and therapeutic approaches.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Organismal aging and age-associated diseases, including many cancers, are driven by the slow accumulation of damage in cells and tissues over time. In some instances, accumulation of deleterious mutations increases genomic instability in dividing cells and disrupts the proliferation and regenerative capacity of adult stem cells that maintain tissue function. Repeated injuries can also stimulate wound- induced inflammation that accelerates aging across an entire organism. Interventions that increase regenerative capacity of injured and aging tissues have the potential to improve quality of live and disease outcomes. However, they require an ability to both detect and rejuvenate aging tissues without increasing risk of disease. While researchers have discovered genes and signaling pathways that can expand life span in short-lived organisms like mice, flies, nematodes, and yeast, there are relatively few studies of anti-aging mechanisms in regenerative organisms with resistance to age-associated disease. This proposal exploits a highly regenerative flatworm, Schmidtea mediterranea, to address this gap. Asexual S. mediterranea undergo indefinite repeated cycles of clonal expansion through asexual reproduction and whole-body regeneration, but do not outwardly appear to suffer from age-associated tissue degradation. We have discovered that planarian tissues do accumulate age-associated damage during growth, but this damage does not result in stem cell exhaustion or a decline in regenerative capacity across clonal generations. Therefore, the central goals of this proposal are to (1) determine how age-associated damage manifests in asexual planaria throughout the animal lifecycle and (2) Identify the cell types and signaling pathways that regulate cellular aging, detection of aged tissues, and tissue rejuvenation. We will use a combination of well-established assays for conserved aging hallmarks, unbiased genomics and transcriptomics methods, and RNAi gene depletion studies to achieve these goals. Together, our proposed work will produce the most complete functional characterization age-associated signaling in asexual S. mediterranea to date and rigorously establish planaria as a tractable discovery model for anti-aging research. By expanding our understanding of adaptations for anti-aging and whole-organism rejuvenation in planaria, we can build a foundation of knowledge that will ultimately improve anti-cancer and anti-aging therapies in humans.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY / ABSTRACT Bladder conditions such as incontinence and urinary tract infections (UTI) are increasingly common with aging and driven by poorly understood changes that occur at the cellular and molecular levels. The objective of this project is to define the molecular pathways that underpin how aging makes the bladder epithelium (urothelium) prone to UTIs and develop new therapeutics to target those pathways. Data indicate that IFRD1 plays a key role in ribosomal quality control, translation, ER trafficking, and ER stress; and loss of IFRD1 produces aging phenotypes. The central hypothesis is that aging disrupts the cellular function of proteostasis, particularly ribosome dynamics during protein translation, modification, and trafficking between the ER and Golgi. Aim 1 will determine the role of ribosomal and translational quality control machinery in the maintenance of bladder homeostasis in young, aged, or Ifrd1−/− urothelium of mice. Cycloheximide treatment will be used to inhibit translation in young mice, and translation efficiency will be quantified with a puromycinylation assay to test if treatment is sufficient to induce phenotypes of aging or Ifrd1−/− bladders. UTI will be induced using uropathogenic E. coli., and bladder tissue will be collected to assess molecular-cellular and inflammatory responses using histological analysis, western blots, and qRT-PCR to measure expression of ribosome-associated quality control markers and IFRD1 expression. To determine urothelial-intrinsic role, we will use a newly generated state of the art 3D- murine urothelial organoid system, with the potential to extend investigations to a human urothelial organoid system. Aim 2 will determine whether chronic ER stress is sufficient for promoting aging phenotypes, and if alleviating ER stress will ameliorate the phenotypes. In aged, young, and Ifrd1−/− mice bladders and organoids, the expression of ER stress proteins and uroplakins will be compared. Western blots, qRT-PCR, and immunostaining will be used to determine if uroplakin trafficking is altered in aged bladders, as in the Ifrd1−/− phenotype. Inflammation, DNA damage, lysosomes, mitochondria, urothelial cell shedding, and expression of ER stress response proteins will be assessed in response to the ER-stressor tunicamycin and ER stress alleviator TUDCA to determine if this stress drives aging in the urothelium using organoid models. Aim 3 will determine the effects of pharmacological interventions on aging bladder and identify novel proteins governing bladder aging. Understanding the effects and mechanism of action in these drugs will establish a foundation on which translational research can work to develop novel interventions for the treatment of urinary tract conditions that are known to progress with age.

NIH Research Projects · FY 2025 · 2024-09

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that is usually asymptomatic at an early stage. In addition, metastases can be present even when the cancer size is relatively small. Most PDAC patients succumb within 6 months of diagnosis with only 12% of patients surviving more than five years. Clinical assays that can assist detection of incipient and early PDAC when effective treatment is possible would improve the survival rate and change the grim outlook of this disease. Unfortunately, CA19-9, the current blood-based clinical biomarker for PDAC, does not provide the accuracy needed for diagnosis, even in high-risk groups of patients. The demands on an effective biomarker for PDAC detection are considerably high compared to many other cancer biomarkers, because while the disease is less common, it is more deadly. Mass spectrometry (MS)-based methods have increasingly been emerging for clinical diagnosis, representing a technology ripe for cancer biomarker detection in clinical settings. We have previously developed a MS-based proteomic assay that significantly outperforms CA19-9 for PDAC blood detection. In this study, we aim to further validate this existing assay for PDAC blood detection through collaborative, multi-discipline efforts. Our team will conduct in-depth analytical and clinical validations to: 1) determine the performance characteristics and robustness of the assay in the context of clinical utilization; 2) refine the assay accuracy and thresholds for PDAC detection using well-annotated case-control cohorts from multiple centers; and 3) evaluate the efficacy of the assay for early detection of PDAC using pre-diagnostic cohort. The development of this proteomic assay will provide a non-invasive and affordable blood test to assist current work-up for PDAC detection in high-risk populations. This study is well-aligned with NCI’s Special Interest in analytical and clinical validation of assays for early detection of cancer using existing cohorts and prospective study designs.

NIH Research Projects · FY 2024 · 2024-09

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease that is usually asymptomatic at an early stage. In addition, metastases can be present even when the cancer size is relatively small. Most PDAC patients succumb within 6 months of diagnosis with only 12% of patients surviving more than five years. Clinical assays that can assist detection of incipient and early PDAC when effective treatment is possible would improve the survival rate and change the grim outlook of this disease. Unfortunately, CA19-9, the current blood-based clinical biomarker for PDAC, does not provide the accuracy needed for diagnosis, even in high-risk groups of patients. The demands on an effective biomarker for PDAC detection are considerably high compared to many other cancer biomarkers, because while the disease is less common, it is more deadly. Mass spectrometry (MS)-based methods have increasingly been emerging for clinical diagnosis, representing a technology ripe for cancer biomarker detection in clinical settings. We have previously developed a MS-based proteomic assay that significantly outperforms CA19-9 for PDAC blood detection. In this study, we aim to further validate this existing assay for PDAC blood detection through collaborative, multi-discipline efforts. Our team will conduct in-depth analytical and clinical validations to: 1) determine the performance characteristics and robustness of the assay in the context of clinical utilization; 2) refine the assay accuracy and thresholds for PDAC detection using well-annotated case-control cohorts from multiple centers; and 3) evaluate the efficacy of the assay for early detection of PDAC using pre-diagnostic cohort. The development of this proteomic assay will provide a non-invasive and affordable blood test to assist current work-up for PDAC detection in high-risk populations. This study is well-aligned with NCI’s Special Interest in analytical and clinical validation of assays for early detection of cancer using existing cohorts and prospective study designs.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract To conquer cancer, researchers with diverse backgrounds and expertise must work together to discover molecular targets and develop therapeutics. Many investigators are unable to accomplish their research goals without access to specialized expertise, sophisticated technologies, and/or expensive instrumentation that are impossible to master or operate in their own labs. The members of the P30CA125123 grant-supported Dan L Duncan Comprehensive Cancer Center (DLDCCC) at BCM are funded with more than $180M cancer research grants with over $41M NCI grants (direct costs). Dr. Reddy, a highly accomplished cancer physician scientist, is the Program Director of the P30 grant, the director of the DLDCCC, and the Unit Director of this R50 application. As a Genetically Engineered Rodent Models (GERM) core-based non-tenure track research specialist (RS) and the Technical Director of the GERM core, I devote >70% of my effort to play an irreplaceable role in providing comprehensive mouse cancer model services to these NCI-funded cancer research programs with my specialty and our GERM core’s state-of-the-art equipment. These services include: generation of various genetically engineered mouse (GEM) models by the CRISPR/Cas9 system using zygotes, the gene-targeting system using embryonic stem (ES) cells, or the microinjection of transgenes into zygotes; in vitro fertilization and embryo transfer to rederive and/or quickly expand GEM models; cryopreservation of sperm and embryos for long-term storage of GEM lines; and genotype analysis of GEM lines. In the past 5 years, I and my team members have generated 1653 knockout, knock-in and transgenic GEM models, many of which are essential for the success of many NCI-funded cancer research programs. I am a highly experienced cancer researcher who has published over 80 manuscripts. I have mastered methodologies and hold specialized expertise necessary for mouse and rat embryo manipulation and genome engineering. I am a core leader who has contributed significantly to NCI- funded cancer research programs led by other investigators. Funding of this R50 application will provide me with a higher degree of autonomy to develop new technologies and services. In collaboration with investigators, I will develop new embryo manipulation and genome editing systems for rat and Nile rat, as well as new genome- editing methods/strategies for mouse, such as prime editing to minimize off-targeting events, and approaches to increase conditional allele production efficiency and conditionally express disease-associated variants. I will implement these new systems and technologies into our core services to better serve cancer researchers with novel rodent cancer models. With the R50 support, I will update my knowledge and ensure the usage of the best genome-editing system and methodology to generate rodent models by attending meetings and interacting with other leaders in the field. My goal is to make our GERM core fully capable of providing comprehensive services of mouse, rat and Nile rat cancer models to support cancer research, and develop my career into an internationally recognized RS in the field of rodent genome editing and rodent cancer models.

NIH Research Projects · FY 2025 · 2024-09

Pulmonary hypertension (PH) represents a grave and relentless medical condition, characterized clinically by elevated pulmonary arterial pressure and augmented pulmonary vascular resistance (PVR). This pathophysiological state inevitably progresses to the development of right ventricular (RV) hypertrophy (RVH), culminating in RV failure, and ultimately, mortality. Moreover, PH could be a confounding complication of chronic lung pathology particularly secondary to interstitial lung disease (ILD), including its severe type- Idiopathic pulmonary fibrosis (IPF). The primary driver of increased PVR in ILD-PH, similarly as in other PH subtypes, is pulmonary vascular remodeling, a complex process involving various cells in pulmonary arterial wall. However, the progression of PH from ILD remains largely unknown. Recently, the first inhaled vasoactive drug-treprostinil was approved for treating ILD-PH. However, concerns about its potential side effects and high costs highlight the urgent need for innovative strategies to manage ILD-PH. Unfortunately, there is currently no treatment specifically designed to target vascular remodeling in ILD-PH. Bone morphogenetic protein (BMP)-binding endothelial regulator (BMPER) is highly expressed in the lung; however, our understanding about its role in lung diseases is very limited. BMPER is a secreted regulator that regulates vascular development via fine-tuning the BMP pathway. BMP plays a pivotal role in the development of pulmonary arterial hypertension. Animal models targeting BMP pathway components (i.e. BMPR2, Gremlin1) also illuminate their regulatory functions in other forms of PH, including that associated with ILD. However, the role of BMPER has not been studied in any forms of PH. In this study, we will define a new and lung-specific paracrine regulatory mechanism mediated via BMPER for vascular remodeling during the progression of ILD-PH. First, we will define the transcriptional regulation of BMPER during the progression of ILD-PH. Next, we will establish mechanisms governing BMPER regulation of pulmonary vascular remodeling. Last, we will assess the tissue-specific BMPER expression in the development of vasculopathy in patients with ILD-PH.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Observational learning is a type of social learning and a fundamental cognitive behavior in humans and social animals. How neural circuits in the brain enable this type of learning is not understood. Utilizing an observational spatial working memory task, where an observer rat learns to first observe the spatial trajectory of a demonstrator rat and then run the trajectory itself for rewards, this project studies the behavioral and neural circuit processes underlying reward-directed observational learning. We will focus on a neural circuit hypothesis that interactions between the hippocampus (HP) and the anterior cingulate cortex (ACC) acquire a vicarious reward signal during observation and maintain a plan afterward for future spatial trajectories. By conducting behavioral testing, simultaneous high-density tetrode recording, and closed-loop electrical and optogenetic manipulations in animals performing the observational spatial working memory task, we will determine key behavioral factors involved, underlying neural activity patterns and their interactions in HP and ACC, and how behavioral performances are altered by time- and activity-specific disruptions of HP or ACC neurons. The outcomes of this study will significantly advance our understanding of behavioral and neural mechanisms in observational learning. The neural circuit knowledge produced and the manipulations tested in this project may generate novel insights into how observational leaning is impaired in psychiatric disorders.

NIH Research Projects · FY 2026 · 2024-09

PROJECT SUMMARY The cingulum bundle (CB) is a major white matter fiber system mediating connectivity of the affective network. As mood monitoring and regulation are controlled by the affective network, the CB is often investigated in relation to the pathophysiology and treatment interventions for mood disorders. Several efforts have attempted to characterize the CB relative to mood disorders, however, previous approaches have overlooked consideration of interindividual variability in local anatomy. The most common anatomical variation in the cingulate region is the presence or absence of paracingulate sulcus (PCS) morphological pattern. Despite 70% of the population exhibiting a distinct PCS in at least one cerebral hemisphere, and its presence inducing known reorganization and connectivity changes in cingulate gray matter, its influence on white matter remains unknown. Interindividual variability in paracingulate sulcus (PCS) morphology may alter local white matter organization, specifically the cingulum bundle (CB) fiber system. Given the importance of the CB in mediating affective network connectivity implicated in mood disorders and treatment efforts, characterization of structural and functional implications of PCS variability is necessary. The goal of this project is to identify CB connectivity alterations across PCS morphology and characterize ill-defined PCS-related white matter with respect to the cingulum fiber system. I hypothesize that PCS morphology will influence CB connectivity with affective network regions and that the PCS-related white matter, termed paracingulate white matter (pcWM), will demonstrate affective network connectivity. I will evaluate these central hypotheses in the following two Specific Aims. Aim 1. To use high-resolution diffusion-based probabilistic tractography to determine the impact of PCS morphology on CB structural connectivity and characterize pcWM. Aim 2. To employ human intracranial single pulse stimulation directed to the CB to determine the effect of PCS morphology on electrographically-defined effective connectivity of the CB. Completion of these Aims will provide novel insight into the relationship between white matter organization and sulcation pattern. Further, elucidating the influence of PCS pattern on connectivity perturbation of the CB is necessary for informing and optimizing treatment intervention for mood disorders like depression. More than 30% of patients with Major Depressive Disorder (MDD) suffer from suboptimal responses and are diagnosed with Treatment Resistant Depression (TRD). With a continued lack of response to other methods, patients may undergo an invasive surgical approach called deep brain stimulation (DBS) for depression treatment. DBS for TRD traditionally targets a confluence of white matter paths, and despite inconsistent response, CB engagement is the primary predictor of optimal patient response. Parsing the correspondence of PCS pattern and the connectivity of the CB may reveal a contributing factor to response inconsistency, thereby informing neurosurgical planning and improving the capacity for predicting treatment response in TRD for DBS.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY As the site of action potential initiation, the axon initial segment (AIS) contains high densities of voltage-gated sodium (Nav) and potassium (Kv) channels that generate and shape the action potential. Kv1 channels are a prominent type of ion channel at the AIS that modulate action potential waveform. Despite their functional importance, the mechanisms responsible for the localization of these channels at the AIS are poorly understood. In contrast, the recruitment of other ion channels to the AIS is well established and depends on interaction with the AIS scaffold protein, AnkyrinG (AnkG). Nav1 and Kv7 channels directly interact with AnkG through a conserved binding motif that Kv1 channels lack. However, unlike Nav1 and Kv7 channels, Kv1 channels contain PDZ binding motifs, which permit interactions with PDZ domain containing proteins. Kv1 channels have been shown to interact with the PDZ domain-containing scaffold protein PSD93 that is highly enriched at the AIS. Although in vitro knockdown studies found that PSD93 is required for Kv1 channel clustering at the AIS, subsequent in vivo knockout studies demonstrated that PSD93 is dispensable, suggesting that other mechanisms can cluster Kv1 channels at the AIS. Previous work also suggests that AnkG is required for AIS Kv1 channel clustering, but the link between AnkG and Kv1 channels is unknown and may be indirect. Thus, the mechanisms underlying Kv1 channel localization at the AIS are unclear. Our lab recently identified a new AIS scaffold protein, SCRIB, that directly binds AnkG. SCRIB contains multiple PDZ domains and may constitute a novel mechanism for clustering Kv1 channels at the AIS. However, the function of SCRIB at the AIS has not been investigated. The objective of this proposal is to define the molecular mechanisms responsible for clustering Kv1 channels at the AIS. Aim 1 will use AAV- and CRISPR-based knockouts, conditional knockout mouse models, and immunostaining to determine if SCRIB, PSD93, and AnkG are necessary for AIS Kv1 channel clustering in vitro and in vivo. Aim 2 will use co-immunoprecipitations and surface clustering assays in heterologous cells to determine the molecular interactions between Kv1 channels and the AIS scaffold proteins SCRIB, PSD93, and AnkG. The proposed experiments will establish a model for how Kv1 channels are localized at the AIS. The Rasband lab has extensive experience elucidating the molecular mechanisms underlying AIS assembly. Thus, the proposed experiments are designed to take advantage of this expertise and the many tools and reagents available in the Rasband lab. This research project will also provide a robust training experience for my development as a molecular neurobiologist. Through the completion of the proposed aims, I will advance the research skills, critical thinking skills, scientific communication skills, and mentorship skills that I will need for my future career as an independent investigator.

NIH Research Projects · FY 2024 · 2024-09

ABSTRACT The ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) system consisting of a SCIEX QTRAP® 7500 MS System and Shimadzu Nexera Series 40 UHPLC system, contained in this proposal, represents the most innovative LC/MS-based instrumental platform for small-molecule quantitative bioanalysis on the market. At present, NIH-funded PIs working across a number of Texas Children’s Hospital (TCH) Departments are increasing their application of quantitative mass spectrometry (MS)-based bioanalysis in support of their translational research programs in pediatric medicine. These Departments include Pediatrics-Gastroenterology, Hepatology, and Nutrition; Nephrology; Cardiology; Obstetrics and Gynecology; and, Laboratory Medicine. Currently, the Texas Children’s Microbiome Center (TCMC) is the only TCH-based facility, in the entire Texas Medical Center (TMC) campus that possesses UHPLC-MS/MS instrumentation and expertise. The TCMC – Metabolomics and Proteomics Mass Spectrometry Laboratory (TCMC-MPMSL) has witnessed steady growth in sample volume from 2020 to 2023, and our single UHPLC-MS/MS system is currently analyzing ~9,000 samples per annum while consuming ~90% of the available instrument capacity. The reasons for our funding request are two-fold: 1) expand our bioanalytical capacity to support the increasing levels of pediatric-based translational research being performed at TCH; and, 2) upgrade our instrumental capabilities to drive new biomedically-relevant discovery in support of pediatric patient care at TCH. Altogether, the instrument included in this proposal will provide much needed capacity and new capabilities that the TCMC-MPMSL can leverage to advance the basic and clinical research efforts of NIH-funded investigators at TCH in pediatric medicine.

NIH Research Projects · FY 2025 · 2024-09

The K12 Companion Application for our CSTA, entitled “Consortium for Translational and Precision Health (CTPH)”, seeks to promote the career development of early-career faculty members to become leaders in translational science, with an emphasis on precision health. Our K12 program capitalizes on the strengths of two institutions: Baylor College of Medicine (BCM) and the University of Houston (UH). BCM is a top-tier academic health sciences center with a rich history of innovative discovery leading to human applications in genetics, biotechnology, medical research and medicine. UH is a Tier-1, comprehensive, and vibrant research university with prestigious programs in pharmacy, computer science, business, mathematics, behavioral science, population health, and biomedical engineering. Our program will promote synergy among the partner institutions to create an integrated and thriving environment for developing the next generation of experts in translational science. Their geographic proximity in Houston facilitates the partnership between BCM and UH, only 4 miles apart, in one the nation’s fastest growing cities. Our K12 will support physician-scientist and PhD scholars for a two-year training program with an optional third year, with 75% protected time devoted to mentored research and career development activities. We will recruit scholars from multiple disciplines and provide a cross-disciplinary, team-based training program that integrates competencies in translational science. Our scholars will receive training in precision health and relevant disciplines, including digital health promotion, artificial intelligence and machine learning, determinants of health, implementation science, bioinformatics, multiomics, leadership, and related concepts. Scholars will enroll in a unique curriculum consisting of 1) a common core team-based curriculum, 2) an individualized curriculum, and 3) experiential activities. Our K12 program provides an ideal opportunity to enhance the pool of highly qualified, multidisciplinary faculty who contribute to the health of Greater Houston communities through innovative translational science.

NIH Research Projects · FY 2026 · 2024-09

ABSTRACT Neural tube defects (NTDs), including spina bifida (SB), have a devastating impact on the health and development of infants and children, with economic, social, and physical demands or hardships placed on caregivers. SB affects approximately 2,500 live births per year in the United States, with environmental and genetic factors playing a role in its etiology. By combining bioengineering and placental-derived stem cell approaches, the CuRe (Cellular Therapy for In Utero Repair of Myelomeningocele) trial has recently advanced surgical effectiveness and improved clinical outcomes. However, in utero repair requiring fetal exposure at mid- pregnancy to reduce the ongoing damage of the exposed spinal cord represents an invasive procedure. By leveraging our expertise in the synthesis of biomimetic therapeutic strategies able to induce tissue repair by inducing a regenerative cascade at the site of lesion and in the generation of genetically induced model systems, this project aims to devise less invasive, novel intervention strategies for endogenous in utero repair of SB. The overall hypothesis is that by creating a pro-regenerative environment within the amniotic cavity, it is possible to activate the cellular and molecular cascades required to reduce the severity of SB lesions. To test these hypotheses, we will i) determine the therapeutic efficacy of amniotic fluid-based cell free strategies to modulate the in utero environment and reduce the severity of SB in a clinically relevant in-house mouse model (Fbpk8 knockout mice) (Aim 1) and ii) evaluate the protective and regenerative potential of a biomimetic thermogel for in utero repair of SB lesions in mouse and rabbit models (Aim 2). The proposed research is expected to enhance our understanding with respect to the impact of biomimetic strategies inducive of regeneration on the development of neural tube tissues following genetic and mechanical disruption. The implications of these therapeutic intervention strategies are timely and critical, as stem cell-based, and bioengineering approaches are already being deployed clinically in other areas, but a minimally invasive, early in utero intervention is still needed for families and infants affected by SB.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Nonribosomal peptides (NRPs) are an abundant class of microbial natural products: small molecules with evolved chemical structures and biological activities that make them an essential source of pharmaceuticals. NRPs have served as leads for antifungal, anticancer, and immunosuppressant drugs, but are particularly important for antibacterial therapeutics, with over 40% of approved antibiotic classes being NRPs or derivatives. The diverse structures and activities of NRPs are generated by a conserved class of proteins known as nonribosomal peptide synthetases (NRPSs), which are composed of multi-domain modules that progressively build NRPs as enzymatic assembly-lines. As the composition and sequence of domains within each module determine which of >500 known monomers will be incorporated into the growing NRP, NRPS engineering has been viewed as a promising approach for producing designer molecules in live cells. However, while ribosomal peptides can be reprogrammed through the genetic code, NRP sequences are determined by the “adenylation (A) domain code”, which describes a series of conserved residues and positions within the amino acyl binding pocket. Unlike the genetic code, the A-domain code is not directly programmable, and attempts to re-write this code and which amino acid is activated by a given domain generally results in poorly active enzymes. Transplantation of binding pockets can occasionally yield domains and modules with desired specificity, but this often fails and still relies on access to characterized protein donors. Directed evolution is a promising approach for re-coding NRPSs, but current methods are slow, manual, and bespoke solutions that rely on specific activities from the resulting natural product or on reactive chemical handles. In this proposal, we will develop a plug-and- play platform for the continuous directed evolution of NRPS activity and substrate specificity. This approach uses promiscuous NRPS components known as epimerization domains and type II thioesterase domains to convert amino acids selected by A domains into (D) isomers. As (D) amino acids (D-AAs) are not present in the cell, they can be selectively detected and quantified by natural D-AA transcription factors, linking NRPS activity and specificity directly to gene expression. In aim 1, we will demonstrate the generation of D-AA signals from NRPS modules and optimize D-AA transcription factors, enabling live cell reporting for NRPS substrate specificity. In aim 2, we will use this system to drive phage-assisted continuous evolution (PACE) of NRPS modules, providing a rapid and adaptable means of evolving highly functional enzymes with desired substrate specificities. This proposal will deliver the first live-cell reporter system for NRPS substrate specificity and enable the rapid evolution of NRPS components with designer specificities. These advances will unlock our ability to program these pharmaceutical assembly-lines for on-demand production of designer molecules in live cells.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY A group of eleven NIH-funded investigators requests funding for the purchase of a Standard Biotools CyTOF XT mass cytometer with the Hyperion XTi imaging platform, for installation in the Cytometry and Cell Sorting Core at Baylor College of Medicine. A primary application of the instrument will be the analysis of both cell suspensions and tissue sections from human subjects and from rodent models of human diseases. The investigators participating in this application are in two different institutions within the Texas Medical Center and do research in the fields of immunology, cancerology, cardiovascular disease and neuroscience, and are at various stages of their careers in academia. In addition to greatly advancing the individual research programs of the users, the instrument will be available to all investigators at institutes within the Texas Medical Center and in the greater Houston area, fostering collaborative research and providing an important regional resource.

NIH Research Projects · FY 2025 · 2024-09

Over 11,000 infants are diagnosed annually with cerebral palsy (CP) with lifetime medical costs >$1.4 million/person. Elevated muscle tone includes dystonia (involuntary muscular contraction) and spasticity (velocity-dependent muscle stiffness), which often co-exist, greatly impairing function, mobility, and quality of life and causing pain and bony deformities. After decades of research, dystonia remains poorly understood and managed. Intrathecal baclofen (ITB) delivered continuously via an implanted pump is widely used to manage dystonic CP despite equivocal efficacy data and very low evidence supporting ITB use. Co-morbid spasticity is a major confounder that can mask dystonia; further, multiple patterns of brain malformation/injury cause dystonia and most studies fail to address dystonia triggers, e.g., pain. A 2023 study, however, found that despite limited dystonia reduction, ITB improved caregiving ease, pain, comfort, and mobility. This revised proposal specifically seeks to overcome these identified serious limitations of prior studies and thoroughly investigate ITB’s effects using an adequately powered, prospective observational cohort design. This clinical trial will use a standardized titration protocol to optimize ITB dosing. We hypothesize that children with more severe baseline dystonia, spasticity, and pain, as well as those with white matter brain injury patterns, will show the best overall response to ITB. AIM 1) Determine the 12-month effects of ITB on a cohort of 65 clinically representative, ITB naïve, children with dystonic CP using a standardized titration protocol and a battery of assessments to capture critical components of the child’s function and well-being. AIM 2) Complete a detailed characterization of brain malformation and injury patterns in children with dystonic CP. This will be the first study to combine multiple imaging analysis techniques in this population, including the MRI classification scale for CP, lesion network mapping, and tract based spatial statistics. AIM 3) Explore potential differences in outcome patterns as related to patient characteristics and use findings to guide the development of a highly sensitive multidimensional or composite measure that captures clinically important areas of change for the child and family. This revised study builds on new preliminary data and capitalizes on the expertise of an established transdisciplinary team, a high-resource academic clinical environment, and a diverse Parent-Patient Council. The study’s findings are likely to have high impact to improve future treatment of children with dystonic CP, including identifying key patient characteristics associated with more or less positive outcomes and developing a sensitive, efficient multidimensional composite measure to assess treatment responsiveness.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Adolescence is most strikingly characterized by the pubertal growth spurt. It is also a time when the prevalence of depressive and anxiety disorders increases dramatically, a fact reflected in the widespread use of antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs). In fact, antidepressants comprise the second to third most commonly prescribed medication class in this age group. We and others have found that SSRIs are associated with height growth suppression in adolescents. This was particularly true in boys undergoing puberty. To build on these findings, we recently completed another study (R21HD097776) of children and adolescents undergoing puberty and starting treatment with one of two commonly prescribed SSRIs, sertraline and fluoxetine. We again found that, over the 6-month follow-up period, the higher the SSRI dose, the more significant height growth suppression was. In fact, SSRI use reduced growth by about 50% of that observed in unmedicated participants. Notably, sertraline was associated with the most significant deleterious effect on height. Moreover, the higher the SSRI dose, the lower the serum concentration level of insulin growth factor 1 (IGF-1), the principal marker of growth hormone (GH) neurosecretory function. To better characterize the clinical implications of these findings, we now propose to 1) determine whether the suppression of height growth and IGF-1 plateaus or continues unabated over the course of a 2-year follow- up, 2) whether differences between the 4 most prescribed SSRIs (sertraline, fluoxetine, citalopram, and escitalopram) exist, and 3) whether SSRI treatment merely slows growth down or, rather, stunts it, thus reducing anticipated adult height. The latter will be evaluated using serial assessments of bone age. In sum, the proposed study will be the first to investigate the magnitude of height growth suppression induced by SSRIs, its clinical predictors, and its long-term sequelae, shedding light on a currently little-recognized side effect of a widely and increasingly used medication class. The information will be critical to informing clinical decision making.

NIH Research Projects · FY 2025 · 2024-09

The Consortium for Translational and Precision Health (CTPH) will serve as a unique bi-institutional hub for infrastructure, services, community engagement, and workforce development to advance clinical translational science (CTS). Led by Baylor College of Medicine (BCM) and the University of Houston (UH), the overall mission of CTPH is to foster research and innovations across the translational spectrum to advance precision health in partnership with local and regional stakeholders. CTSA funding will enrich the CTPH’s ability to launch new initiatives, enhance our existing efforts, and effectively disseminate discoveries and innovations to populations in Houston and the among the CTSA network. Building on our current high-quality programs, we will develop an integrated, adaptable, and sustainable clinical and translational research infrastructure providing superior, accessible resources and services, extending value to all stakeholders. We propose the following overarching aims: Aim 1. Develop, demonstrate, and disseminate new technologies, platforms, and research methods that advance innovations in precision health. We will support precision health initiatives by developing robust platforms to advance translational science and accelerate transformative biomedical, ‘omics, clinical, and population health research. Collaborative, interdisciplinary teams are poised to establish novel lines of research in CTS and precision health, and bolster digital approaches for continuous health screening, monitoring, and tailored interventions. Aim 2. Reach and engage broad populations and across the lifespan to reduce health challenges and improve population health. Our deep commitment to reduce local and regional health challenges will be strengthened through increasing engagement with populations served by the CTSA, incorporating community perspectives in research across the lifespan, and reducing institutional barriers to full participation of community partners in all aspects of the CTPH. Aim 3. Educate, train, and sustain a robust translational workforce to improve the efficiency and generalizability of clinical and translational research and support a pipeline of new researchers. We will build innovative training and career development programs across the translational workforce to drive change, transform science, and significantly improve population health. Aim 4. Create a culture for clinical translational science teams to collaborate and transform translational workflows. Our programs will simplify access to resources, emphasize team science, offer joint enrichment programs, and accelerate workflows through improved efficiency and stakeholder-informed perspectives. These initiatives will be implemented by an experienced leadership team with authority over the relevant administrative structures, which will expand synergies in CTS between the largest University system in Houston with the leading biomedical research institution in Texas.

NIH Research Projects · FY 2025 · 2024-09

Inflammatory Bowel Disease (IBD) is a debilitating condition characterized by chronic inflammation, leading to the erosion of essential intestinal functions. The risk of developing IBD, including related diseases like Crohn's and ulcerative colitis, involves a complex interplay of over 250 genetic and environmental factors. Dysbiosis of the gut microbiome and pathogen proliferation often worsen the disease pathophysiology. While some common genetic risk factors, especially those related to the immune system, are well understood, the functional contributions of most risk alleles remain unclear. Moreover, the majority of known genetic risk factors have been identified in European ancestry populations, which may not translate to more diverse populations. Our long-term goal is to gain a comprehensive understanding of the genetic risk factors contributing to IBD development. We hypothesize that genetic alterations in intestinal gene function create conditions for persistent dysbiosis and inflammation, driving IBD. To achieve this, we have established a robust cross- organismal platform for high-throughput functional profiling of IBD candidate genes, advancing our understanding of the disease's mechanisms. We aim to address our hypothesis through two specific aims: 1) conducting high-throughput screening and phenotypic characterization of IBD risk genes in C. elegans, a nematode microbiome model; and 2) validating IBD risk alleles in human intestinal organoids (HIOs). Importantly, our research will encompass and integrate risk alleles from diverse populations, providing a more comprehensive assessment of IBD risk. This study is significant because it delves into the functional profiling of IBD risk genes at the organismal level, a less- explored area. Further, it extends its focus to diverse multi-ancestry cohorts, filling a critical gap in understanding of the disease. The platform developed also has the potential to be used in studying other microbiome-mediated diseases. Ultimately, this work can enhance our understanding of the genetic landscapes of IBD, offering both broad functional testing and specific mechanistic insights, which are vital for the diagnosis and treatment of this prevalent digestive disease.

NIH Research Projects · FY 2025 · 2024-09

Bipolar Disorder (BD) is a recurrent neuropsychiatric disorder characterized by wide fluctuations in mood, energy, and activity. Although the hallmark of BD is mania in BD-I (hypomania in BD-II), depressive episodes dominate the longitudinal course and account for a disproportionate degree of the morbidity and mortality. About 25% of patients with BD meet criteria for treatment-resistant bipolar depression (TRBD). Given the gravity of the illness and paucity of available therapies, there exists a large unmet need for more effective interventions. We propose an early feasibility study to advance the therapy and neurobehavioral monitoring of TRBD with deep brain stimulation (DBS) targeting the ventral capsule/ventral striatum (VC/VS) using a sensing capable device. We will leverage a smart phone-based software platform paired with the device's chronic sensing capability to record local field potentials (LFPs) time-locked with remote, high-density measures of natural behavior (e.g., from wearables). Our goal is to detect relevant behavioral states including both depression and mania and transitions between these states, including high risk mixed features. Real-time, remote detection of affective states via both behavioral and neural data monitoring will enable new interventional strategies to shift behavior away from pathological behavioral states towards healthier ones, contributing to the long-term stability of patients with BD. Aim 1: Efficacy and safety of VC/VS DBS for TRBD will be examined in a 9-month open-label early feasibility study of 10 subjects with BD-I, followed by a 3-month stabilization period, and then blinded discontinuation at month 12 to confirm active vs. sham response. Efficacy outcome will be based on rating scale measures of initial response at 9 months, absence of recurrence of depression during follow-up at 18 months, and management of emergent (hypo)mania. Aim 2: Mania/mixed feature detection. 2a. Given the risks of inducing mania or mixed states, a clinician-facing dashboard will be created and updated every 24 hours displaying raw data for: affect/mood, energy/activity, sleep, speech, and anxiety. Behavioral and physiological data will be derived from multimodal methods (e.g., computer vision, voice recordings, Apple watch, Oura Ring, etc.), in/outside the clinic, to objectively assess changes in key behavioral domains. 2b. Composite measures of mood and energy/activity will be developed using machine learning (and other methods) based upon data from the trial subjects. The clinician will be notified when a state change (e.g., emerging mania) is identified that warrants adjustment of stimulation. Aim 3: Identify neural markers to be used as classifiers for state changes from depression into either manic or mixed states. Development of neural classifiers for (hypo)mania builds upon research from an NIH funded study of VC/VS DBS in subjects with OCD. The outcome of this aim, coupled with aim 2, has implications for advancing DBS programming with the clinician-in-the-loop and possible future development of an adaptive DBS system in BD. Meeting all milestones could also inform studies of DBS in depression with respect to neurobehavioral feedback to drive DBS programming decisions.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD undergo chronic hemolysis which results in the release of large amounts of free heme, the small molecule co-factor of hemoglobin. Free heme levels are elevated in SCD patients and free heme activates many pro-inflammatory pathways. Heme-induced inflammation contributes to the major complications of SCD including acute vaso-occlusion (painful blockages of blood vessels), acute chest syndrome (life-threatening acute lung injury), and chronic pain. Many patients develop these serious complications even while taking existing standard of care therapies. Thus, it is critical to characterize the inflammatory mechanisms that underlie these complications to hasten the development of effective targeted therapies. Our lab identified a key pro-inflammatory protein activated by heme, caspase-4. Once activated, caspase-4 induces IL-1β release and pyroptosis, a form of inflammatory cell death, in macrophages. Circulating IL-1β levels are elevated in patients with SCD and macrophages from patients with SCD release significantly more IL-1β after heme exposure indicating this pathway is upregulated and active in patients. However, it remains unknown how heme activates caspase-4 and how caspase-4 activation contributes to inflammatory complications of SCD. To fill this gap in knowledge and explore the suitability of caspase-4 as a druggable target in SCD, this project will address the following two aims: 1) determine how heme activates caspase-4 through binding interaction studies and mutagenesis and 2) determine the impact of heme-induced caspase-4 activation on inflammatory complications of SCD in a SCD mouse model. The long-term goal of this project is to develop therapeutics to inhibit this pathway and prevent inflammatory complications in patients. The collaborative training environment at Baylor College of Medicine and Texas Children’s Hospital, the cutting-edge core facilities at these institutions, and the many experts in hematology and innate immunity in the Texas Medical Center will strongly support the success of this project. Overall, this project will comprehensively prepare the applicant for a future career as a hematologist-scientist while providing important insights into the underlying pathophysiology of sickle cell disease.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Eswatini was among the first countries to achieve the UNAIDS 95, 95, 95 targets resulting in substantial declines in HIV incidence and TB prevalence. Despite this achievement adolescent girls still have a 1.5% annual incidence of HIV infection and the case detection gap for children with TB is estimated to be well over 60%. This highlights the need for new strategies that complement current approaches to HIV and TB prevention and detection. This study will explore HIV and TB wastewater surveillance as a strategy to reignite the drive toward elimination. These pathogens are well suited to wastewater surveillance due to long infectious periods during which patients are asymptomatic and unlikely to present to health facilities for testing. Wastewater measures of TB and HIV from specific sampling areas will ultimately help target public health community case detection interventions. In the first phase of this project, we will establish the pipeline for wastewater surveillance of HIV and TB in Eswatini, building off our experience at Baylor College of Medicine in Houston. The second phase will validate and quantify detection of HIV and TB from multiple sites, establishing variability, a range of quantification, and correlation between the HIV and TB levels detected in wastewater with hospital epidemiology, public health reporting data, and HIV recency testing data. The third phase will establish whether proven public health interventions to increase case detection and treatment initiation for people with TB and HIV will result in reductions in HIV and TB in wastewater samples from those same communities. If successful, this approach has the potential to be a game changing, cost-effective and highly innovative tool in the fight against HIV and TB. The candidate is ideally suited to lead this grant, having a background of proven success in pioneering wet-bench virology and immunology as well as seven years of experience leading epidemiologic and clinical research in Eswatini. This project merges these two areas of experience, and success will require the ability to introduce and adapt wastewater sampling to new pathogens with an in-depth understanding of HIV and TB transmission, epidemiology, and prevention strategies. The team assembled for this project is further evidence of the candidate's ability to assemble strong collaborative teams, drawing together partners from numerous health and environmental programs in Eswatini and colleagues at Baylor College of Medicine. The data generated from this project has the potential to add precision to HIV and TB prevention and case detection strategies and create a platform for wastewater surveillance of other emerging pathogens in Eswatini. Early pathogen detection systems in low-resource environments will ultimately improve health security globally. This strategy, candidate and partners are perfectly positioned to catalyze the elimination of TB and HIV transmission in Eswatini and set the stage for improved wastewater surveillance systems throughout sub- Saharan Africa.