University Of Texas At Austin

NIH Research Projects · FY 2026 · 2024-02

ABSTRACT The ability to perform dynamic imaging of time-varying physiological processes in small animal models is critically needed to understand the progression of human disease and develop new therapies. Although dynamic imaging methods have been used to evaluate tumor vascular perfusion in small animal models, the available methods typically provide only two-dimensional (2D) spatial imaging, lack the precision needed for quantitative measurements, or suffer from other drawbacks. Photoacoustic computed tomography (PACT) can circumvent the limitations of existing methods and has been recognized as a promising tool for dynamic small animal imaging. By exploiting the optical absorption of hemoglobin or exogeneous contrast agents, dynamic PACT holds great potential for measuring important time-varying biomarkers such as tumor vascular perfusion and oxygenation and improving the assessments of anti-cancer and other therapies. While exciting, current dynamic PACT technologies for small animal imaging still possess several fundamental limitations. Many biological models require true 3D spatial imaging of time-varying physiological processes. However, most of the available dynamic PACT technologies have been designed to rapidly image two-dimensional (2D) slices. While fully 3D PACT imagers are available, most employ a tomographic measurement process in which a gantry containing acoustic transducers is rotated about the animal. This presents unmet challenges for dynamic image reconstruction because only a small number of tomographic views is available to reconstruct each temporal image frame. Moreover, the ability of the available image reconstruction methods to produce quantitatively accurate estimates of the wavelength-dependent optical properties of an object is largely unproven. For dynamic PACT to be established as a transformative preclinical imaging modality, there remains an urgent need for accurate new image reconstruction methods that can be deployed with widely available 3D imagers that use rotating gantries. The broad objective of this project is to directly address these challenges by developing novel and advanced dynamic PACT image reconstruction methods that permit both four-dimensional (4D) imaging (3D space + time) and five-dimensional (5D) multi-spectral imaging (3D space + time + wavelength). This will be game-changing and will enable, for the first time, high-resolution and quantitatively accurate 4D and 5D whole-body PACT imaging of small animal models with widely available PACT imagers that utilize rotating gantries. The specific aims of the project are: Aim 1. To develop 4D image reconstruction methods for dynamic PACT; Aim 2. To develop 5D image reconstruction methods for dynamic PACT; Aim 3. To refine and validate the proposed methods using well-characterized phantoms; Aim 4. To demonstrate and validate the proposed reconstruction methods in in-vivo studies.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Childhood asthma is a major risk factor for lung disease in adulthood, yet what causes some children, but not others, to have a worse disease course, characterized by persistence of disease and decrements in lung function growth, is unclear. Although some genetic factors have been linked to lung function trajectories, much of the variability in disease course remains unexplained, suggesting that environmental factors are likely important in shaping disease course. Several studies have demonstrated that exposure to higher concentrations of particulate matter (PM) leads to worse lung function growth, but the effects of specific PM sources and composition on disease course and lung function growth are largely unknown. Thus, our overarching hypothesis is that exposure to PM from certain sources and of specific compositions predict persistence of asthma severity and decrements in lung function growth. To test this hypothesis, we will conduct a prospective cohort study of 300 multi-ethnic children, the Texas Home Assessment of Asthma and Lung Exposures (TexHALE) study. We will repeatedly measure lung function, asthma severity, and PM exposure (sources, composition, and concentrations) and examine associations between: 1) lung function growth; 2) persistence of disease severity; and 3) biomarkers associated with airway remodeling and lung function decline with PM exposure (sources, composition, and concentrations).The proposed aims will greatly advance our current understanding of the natural history of asthma, which is critical for developing interventions that modify the trajectory of childhood asthma. The proposed work will identify PM sources that are implicated in worse disease course, providing evidence to extend current air pollution regulation to prioritize targeting certain sources. Moreover, the findings will lend insight into racial and ethnic disparities in disease course, the potential contribution of PM exposure to these disparities, and any disparate effects of PM exposure among racial and ethnic minority populations.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract The objective of this study is to identify how a trauma-focused psychotherapy for PTSD, cognitive processing therapy (CPT), alters the function and information processing of the brain during reward learning. Preliminary data indicate abnormal function of the brain’s reward circuit (ventromedial prefrontal cortex, ventral striatum) during reward learning in PTSD. Changes in this circuitry may serve as one mechanism of therapeutic improvement in diminished positive affect symptoms. This is an important area of study, as PTSD diminished positive affect symptoms (e.g., difficulty experiencing positive emotions, diminished interest) are poorly understood neurobiologically and consistently associated with poorer quality of life and worse clinical outcomes. We will assess neural circuitry function and neural encoding (representation in brain signal) of reward-related information parameters with functional magnetic resonance imaging (fMRI). Parameters will be derived from reinforcement learning (RL) computational models of reward task behavior before and after CPT treatment. Preliminary data indicate CPT alters the function and neural encoding of RL information parameters in PTSD, and that changes vary by whether imminent threat is embedded in the reward processing context. This is an ecologically valid approach to studying this construct, as: a) PTSD is characterized by hypervigilance for threat; b) individuals with PTSD often perceive threat in the absence of explicit threat; and c) trauma-focused psychotherapies utilize techniques that may impact both threat and reward-based processes. The rationale is that improved understanding of therapeutic mechanisms may lead to identification of novel treatment targets, which can be utilized to improve treatment outcomes. The central hypothesis is that CPT will alter neural encoding of RL parameters during reward learning variably as a function of threat context. We will recruit 120 individuals with chronic PTSD. Individuals will undergo clinical and fMRI assessment to measure brain function during reward learning with a contextual threat manipulation. RL computational models will facilitate examination of neural encoding under contexts where threat is present or absent. Participants will then be randomized (stratified on presence of comorbid major depressive disorder to ensure equal representation across arms) to receive either immediate, individual CPT treatment or to a delayed treatment condition (N=60 each). Individuals will then repeat clinical and fMRI assessments, and those in delayed treatment will then commence with CPT treatment. It is predicted that CPT will: a) in the absence of threat, enhance neural RL encoding in the ventral striatum and ventromedial prefrontal cortex and decrease neural encoding in the amygdala; and b) under threat (of shock), promote a reduction in ventral striatal/ventromedial prefrontal RL neural encoding and an adaptive, treatment-facilitated increase in dorsolateral prefrontal cortex RL encoding. Outcomes will enhance understanding of trauma-focused psychotherapy mechanisms and may provide novel future treatment targets.

NIH Research Projects · FY 2026 · 2024-02

Project Summary In the initial 5 years of this grant, our group has advanced the field of endocannabinoid therapeutics by discovering novel metabolic and signaling functions for the endocannabinoid biosynthetic enzyme DAGLβ in inflammation and pain response. We developed drug delivery strategies for localizing DAGLβ inhibitors to inflammatory sites for potent pain alleviation in preclinical models. We developed phosphoproteomic, chemoproteomic, and lipidomic capabilities to enable biomarker and drug discovery in pain. This renewal application will extend our preclinical investigations of DAGLβ inhibitors as non-opioid analgesics to include kinase activation as a testable mechanism. We will build on the key biological discoveries, productivity, and technological investment of the first funding period to address key gaps in knowledge to probe how disruption of DAGLβ lipid metabolism activates kinase signal transduction, test whether augmented kinase activation can be achieved using new liposomal formulations of DAGLβ inhibitors and uncover the druggable phosphoproteome in inflammatory and neuropathic pain states. We plan to test our central hypothesis that DAGLβ signals through bioenergetic pathways to alter the macrophage phosphoproteome in local inflammatory responses in pain. An improved understanding of endocannabinoid biosynthetic mechanisms in chronic pain states is important for guiding development of non-addictive, targeted analgesics to address the opioid epidemic.

NIH Research Projects · FY 2026 · 2024-01

SUMMARY: In fish and amphibians the retina is able to fully regenerate from a variety of insults but this ability is progressively lost in higher vertebrates, with modest regeneration in birds, and essentially no regeneration in mammals. Müller glia (MG) are responsible for retinal regeneration and due to the remarkable regenerative capacity of zebrafish, many studies have used this model to uncover the molecular underpinnings of retinal regeneration. In zebrafish, retinal damage first stimulates MG to adopt a gliotic state, but this is transient and they subsequently reprogram into a stem cell-like state, re-enter the cell cycle and generate multipotent neurogenic progenitors that then give rise to new neurons. In mammals, including humans, the initial MG gliotic response to damage is similar to that in zebrafish, but MG fail to reprogram or generate new neurons. Instead, gliotic responses persist and MG proliferate and eventually form a glial scar. Recent studies have demonstrated that ectopic expression of a key pro-regenerative gene identified in zebrafish, ascl1, in MG of the adult mouse retina, along with inhibition of histone deactylase (Hdac) activity, stimulates a MG regenerative response. Regeneration correlated with increased chromatin accessibility at critical pro-regenerative loci and when combined with other published studies, suggest that the epigenetic landscape regulating the expression of pro- regenerative genes during the injury and regenerative responses is a key regulator of the ability of MG to reprogram and stimulate regeneration. Our knowledge of the epigenetic regulation of retinal regeneration is limited and this is a critical knowledge gap in the field. Indeed, given that regenerative responses can be stimulated from normally non-regenerative MG in the mammalian eye, understanding the epigenetic regulation of retinal regeneration could be transformational in supporting the development of new therapeutics aimed at restoring neurons lost to retinal degenerative diseases and retinal injuries. We have focused on the epigenetic regulation of retinal regeneration, using the zebrafish MG reprogramming and regeneration model. We have generated strong preliminary data supporting a critical role for Brd proteins during MG-dependent retinal regeneration and more specifically, a role for Brd4 during MG reprogramming. We have identified genes regulated by Brd activity and developed and validated novel transgenic tools for manipulating Brd activity and Brd-dependent gene function in MG with spatial and temporal precision. We hypothesize that Brd-mediated regulation of gene expression in MG is a key regulator of MG reprogramming and MG-dependent retinal regeneration. Experiments in this proposal test this hypothesis by determining the roles of specific brd proteins during MG-dependent retinal regeneration and identifying the gene regulatory networks in which they function. When completed, the results of this study will be of high impact in identifying and validating Brd-dependent genes that could be useful on their own in stimulating reprogramming or regenerative responses from mammalian MG or, in combination with other factors, enhance reprogramming and regenerative responses from mammalian MG.

NIH Research Projects · FY 2024 · 2023-09

This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions for the overdose epidemic, including opioid and stimulant use disorders. The NIH HEAL Initiative bolsters research across NIH to address the national opioid public health crisis and improve treatment for opioid misuse and addiction. Alongside prevention, treatment, and supply reduction, harm reduction is a cornerstone of the National Drug Control Strategy. However, there is a critical gap in research on the occupational health of Harm Reduction Workers (HRWs), who are exposed to high rates of lifetime and occupational stress and trauma. High rates of unaddressed occupational stress have been shown to have an adverse impact on patient care and are linked to unmet mental health needs, turnover, burnout, and relapse. HRWs need an occupational stress intervention program that addresses both systems and individual occupational challenges while promoting the capacity to engage with the complex demands of delivering safe high-quality harm reduction care to people who use drugs (PWUD). Stress First Aid is a promising intervention that has been implemented with several first responder groups and in healthcare settings, but it requires adaptation to HRWs and it needs to be studied against a control group in a rigorous clinical trial. In the R61 planning phase (Aims 1-4) of this proposal, focus groups comprised of HRWs and leaders from HROs will be convened to adapt the SFA training content and to inform the SFA training delivery methods that will be piloted in a field test (N = 15). Findings will be integrated into a clinical trial protocol. In the R33 phase of this proposal (Aims 5 and 6), we will conduct a cluster-randomized hybrid type I trial (N = 534) testing the effectiveness of participation in an Addiction Technology Transfer Center (ATTC)-facilitated SFA training and learning collaborative support compared to a no treatment control condition on the following primary outcomes: social-support, burnout and use of SFA concepts. Secondary outcomes are secondary traumatic stress, use of mental health care, engagement, and turnover. The long-term goal of our work is to implement a sustainable and effective occupational stress intervention for HRWs nationally in order to strengthen their important role in the substance misuse work force. Key innovations include: 1) use of ATTCs to provide overarching structure to SFA trainings for HRWs in order to strengthen organizational capabilities to implement and to accelerate findings into practice; 2) first ever adaptation of Stress First Aid to unique occupational context and needs of HRWs; 3) use of qualitative methods to co-adapt and co-design Stress First Aid content, training methods and research methods with and for HRWs; 4) potential to enhance trust and engagement with between ATTCs and HRWs which could enhance diffusion of best practices. If effective, widespread adoption of SFA could improve workforce capacity for high quality care for PWUD.

NIH Research Projects · FY 2024 · 2023-09

Project Summary There is an urgent need to identify modifiable risk factors and intervention approaches that would help to prevent or delay the onset of Alzheimer's Disease (AD). In this regard, several researchers propose that prosocial engagement via helping others should be considered a public health intervention for tackling the AD epidemic. This perspective is based on the accumulated evidence demonstrating how formal volunteering confers a wide range of health benefits for the volunteer, in part through influencing neurobiological systems that also influence AD risk. Although recent reviews indicate inconclusive evidence, a small but growing body of correlational evidence for the link between volunteering and cognitive outcomes is corroborated by findings from randomized control trials, such as the Baltimore Experience Corps. However, earlier studies on prosocial helping behaviors in later life mostly focused on formal volunteering, while little is known about the cognitive benefits of informal helping given directly to non-household individuals in the community, which may be more relevant for African and Hispanic Americans with a richer tradition of informally serving their communities. The overarching goal of the project is to uncover robust evidence linking two forms of helping behaviors (i.e., formal volunteering, and informal helping) and better cognitive functioning so as to establish prosocial engagement as a health behavior that could reduce the risk or delay the onset of AD. Taking advantage of the rich longitudinal data from the NIA-funded Health and Retirement Study (1998-2020), we address the following key aims. First, we identify the extent to which helping behaviors promote higher levels of cognitive functioning and slower cognitive decline while paying close attention to potential racial-ethnic differences for the link between the type of helping and cognitive outcomes. We employ the recently-proposed asymmetric modeling approach to accurately estimate the within-person effects of transitioning into (and out of) helping behaviors. Subsequently, we disentangle rapid cognitive decline driven by genetic risk factors for developing AD from normal, age-related changes in cognitive functioning and identify the dose-response of helping needed to alleviate cognitive decline accelerated by high genetic AD risk. Finally, we situate the helping- cognition nexus within the neighborhood context in which helping behaviors often take place and investigate the extent to which helping others buffers detrimental effects of disadvantaged and/or deteriorating neighborhood socioeconomic status, assessed with Census-tract level data. The research team includes a new investigator and four consultants with expertise covering all facets of the proposed project, and thus the team is well-suited to successfully address the innovative set of project aims. The project is expected to yield high reward outcomes not only in terms of contributing to the scientific literature but also for developing strategies for effective, evidence-driven community-based AD intervention programs, an urgent priority of NIA.

NIH Research Projects · FY 2025 · 2023-09

“This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.” Decades of research have established the importance of early mother-infant interactions for lifelong adaptive social emotional functioning, with implications for the development of later problem behaviors including substance use disorder. A history of maternal sensitivity to infant distress – that is, responses that are consistently contingent, nurturing, and appropriate – is thought to allow infants to internalize socioemotional competencies for maintaining states of emotional security. This is reflected in the development of secure mother-infant attachment and self-regulation behaviors. In turn, mother-infant attachment security is predictive of a constellation of behaviors in childhood and adolescence – including parent-child and parent-adolescent relationship quality, childhood internalizing and externalizing behaviors, competence with peers, and school success – each of which independently predict substance abuse and substance use disorder. The objective of this proposal is to advance opportunities for research and preventative interventions for the intergenerational transmission of substance use disorders by developing and validating mobile sensor algorithms that can be used to remotely assess maternal sensitivity in both standardized protocols and everyday ecologically valid home interactions. Using audio recorders worn by infants and mother-infant motion and proximity data, we will develop models that can distinguish sensitive from insensitive maternal responses to infant distress. Critically, we develop our models with “training data” from a diverse sample of families, including families at both high- and low- risk for substance use disorders and who speak both English and Spanish. This will ensure that our tools will benefit the families who need them the most. We envision that future efforts could leverage our algorithms to identify families at greatest need for existing evidence-based interventions to improve maternal sensitivity and child outcomes. Once trained, our algorithms could be integrated into “just in time” interventions to provide real-time feedback and progress reports for mothers participating in interventions. Additionally, these algorithms will be invaluable to research examining the development of challenges in early caregiving and how such challenges can become amplified over time. For example, these tools could be used to observe the role of difficult infant characteristics, like aversive or excessive crying, maternal stressors or substance use cravings, and maternal support systems, including paternal involvement. As such, the innovative tools produced in the present proposal will both contribute to real-world public health efforts and expand research on the dynamics of early child development.

NIH Research Projects · FY 2026 · 2023-09

This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions for the overdose epidemic, including opioid and stimulant use disorders. The NIH HEAL Initiative bolsters research across NIH to address the national opioid public health crisis and improve treatment for opioid misuse and addiction. Alongside prevention, treatment, and supply reduction, harm reduction is a cornerstone of the National Drug Control Strategy. However, there is a critical gap in research on the occupational health of Harm Reduction Workers (HRWs), who are exposed to high rates of lifetime and occupational stress and trauma. High rates of unaddressed occupational stress have been shown to have an adverse impact on patient care and are linked to unmet mental health needs, turnover, burnout, and relapse. HRWs need an occupational stress intervention program that addresses both systems and individual occupational challenges while promoting the capacity to engage with the complex demands of delivering safe high-quality harm reduction care to people who use drugs (PWUD). Stress First Aid is a promising intervention that has been implemented with several first responder groups and in healthcare settings, but it requires adaptation to HRWs and it needs to be studied against a control group in a rigorous clinical trial. In the R61 planning phase (Aims 1-4) of this proposal, focus groups comprised of HRWs and leaders from HROs will be convened to adapt the SFA training content and to inform the SFA training delivery methods that will be piloted in a field test (N = 15). Findings will be integrated into a clinical trial protocol. In the R33 phase of this proposal (Aims 5 and 6), we will conduct a cluster-randomized hybrid type I trial (N = 534) testing the effectiveness of participation in an Addiction Technology Transfer Center (ATTC)-facilitated SFA training and learning collaborative support compared to a no treatment control condition on the following primary outcomes: social-support, burnout and use of SFA concepts. Secondary outcomes are secondary traumatic stress, use of mental health care, engagement, and turnover. The long-term goal of our work is to implement a sustainable and effective occupational stress intervention for HRWs nationally in order to strengthen their important role in the substance misuse work force. Key innovations include: 1) use of ATTCs to provide overarching structure to SFA trainings for HRWs in order to strengthen organizational capabilities to implement and to accelerate findings into practice; 2) first ever adaptation of Stress First Aid to unique occupational context and needs of HRWs; 3) use of qualitative methods to co-adapt and co-design Stress First Aid content, training methods and research methods with and for HRWs; 4) potential to enhance trust and engagement with between ATTCs and HRWs which could enhance diffusion of best practices. If effective, widespread adoption of SFA could improve workforce capacity for high quality care for PWUD.

NIH Research Projects · FY 2024 · 2023-09

Project Abstract Current treatments of Alcohol Use Disorder (AUD) have shown moderate success in reducing heavy alcohol drinking but are marred with the problem of treatment adherence. In the clinic, opiate receptor pharmaceutics are often combined with cognitive behavioral therapies to improve long-term abstinence. These findings suggest an important relation between endogenous opioid signaling and cognitive factors in the treatment of AUD; however, lapses in treatment can quickly reinstate alcohol drinking, highlighting a knowledge gap in our understanding of how these mechanisms may influence long-term misuse. In this regard, alcohol consumption stimulates the release of endogenous opioids, including enkephalins that bind to mu-type opioid receptors (MOR) commonly found in limbic areas of the brain. The manipulation of MOR signaling alters the rewarding properties of alcohol, with agonists facilitating reward and consumption, and antagonists blocking these responses. The shared relation between opioidergic responses that underlie motivation and cognition are not well understood but present a focal point for addressing complex pathologies that may underlie alcohol-related sensitivities. In this regard, MORs are found in frontal cortical regions that modulate cognitive function, such as the medial prefrontal cortex (mPFC). Preclinical studies in our laboratory demonstrate that alcohol dependence in rats decreases the phosphorylation of MOR in the mPFC, and increases expression of the neuropeptide precursor, proenkephalin (PENK). This pattern of changes overlaps with clinical observations of MOR desensitization in AUD patients. The findings suggest that dependence may dysregulate opioidergic signaling in the mPFC, although the extent to which such conditions surmise changes in the endogenous ligands is unclear. A better understanding is warranted given that adherence to opiate antagonists diminishes over protracted abstinence and may be undermined by molecular intermediates in the processing of small-opioid peptides. Here, we will explore the central hypothesis that non-canonical PENK signaling in the mPFC plays a pivotal role in dysregulating cognitive function during abstinence. Towards this goal, discovery-based and quantitative mass spectrometry approaches will be combined with in vivo microdialysis to broadly capture PENK-mediated signaling in alcohol-dependent rats undergoing abstinence (Aim 1). We will then explore the functional relevance of non-canonical PENK signaling in an operant model of cognitive flexibility, and further determine whether similar dysfunctions in dependence are modulated by mPFC MOR (Aim 2). The results are expected to provide insight into druggable targets that extend beyond conventional opioidergic signaling processes and will lay the foundation for mechanistic studies exploring the role of non-canonical PENK signaling in driving vulnerability to addiction behavior.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The notochord is a highly conserved developmental tissue that extends along the anterior-posterior axis of all chordates, including humans. It is composed of inner vacuolated cells surrounded by an external layer of sheath cells that secrete a thick extracellular matrix. Inflation of the vacuolated cells within the restrictive sheath creates a pressurized rod that supports locomotion in chordates and ultimately patterns the spine of vertebrates. As such, the development of the notochord and spine are intimately linked, and defects in the formation of notochord cells have been linked to scoliosis and vertebral malformations. The notochord is a difficult tissue to study in mouse models since it is already replaced by the spine at the time of birth. In contrast, the external development and optical transparency of zebrafish make them suitable for investigating processes involved in notochord development and maturation. This proposal will use quantitative image analysis, zebrafish genetics, and modern proteomics approaches to define the role of the inositol polyphosphate phosphatase-like 1a (inppl1a) gene in notochord and spine development. Mutations in this gene cause early notochord defects and thoracic scoliosis in zebrafish. In this fellowship proposal, I will test the hypothesis that inppl1a regulates notochord vacuole inflation and, ultimately, the mechanical stability of the notochord with three Specific Aims. In Aim 1, I will determine the role of inppl1a in notochord vacuolation by quantifying changes in notochord cell size and vacuole inflation (1.1) and internal vacuole membrane dynamics (1.2). I will also define the temporal and spatial requirement of inppl1a during notochord development using pharmacological and molecular-genetic approaches (1.3). In Aim 2, I will evaluate the mechanical properties of inppl1a mutant notochords by manipulating mechanical stress (2.1) and vertebral bone mineralization (2.2-2.3) during development. Finally, in Aim 3, I will define the protein interactors of Inppl1a in notochord and spine development. I will use a candidate gene approach (3.1) and a proximity-dependent labeling strategy (3.2) to identify additional proteins required for Inppl1a-dependent notochord vacuole inflation. To supplement these approaches, I will also use modern proteomics techniques to build a comprehensive atlas of the notochord protein interaction network (3.3). In doing so, I will build an invaluable resource for future investigation of proteins involved in notochord development. Altogether, the work in this proposal will add to the knowledge of how notochord cells vacuolate and will ultimately benefit our understanding of human skeletal health and disease. Although the notochord is considered an embryonic tissue, it has been implicated in adult diseases, including intervertebral disc degeneration and chordoma. Additionally, mutations in INPPL1 cause the rare endochondral bone disorder, Opsismodysplasia. Therefore, this work in zebrafish will be significant because it will likely reveal a conserved role for inppl1a/INPPL1 in skeletal development and disease.

NIH Research Projects · FY 2024 · 2023-09

Next generation sequencing and other -omics technologies have spurred the development of precision medicine, but this field is still in its infancy for alcohol use disorder (AUD). Transcriptomic studies have established that alcohol use causes widespread changes in brain gene expression. Brain gene expression profiles can identify alcohol-dependent human subjects and mice and can be used to repurpose pharmaceuticals that reduce excessive alcohol consumption in rodents. However, it is not possible to obtain brain samples from living patients, which limits the translational potential of this approach. Routine blood testing has long been a part of medical care. Blood genomic profiles could potentially be used to non-invasively determine whether a patient is at risk for AUD, provide data-driven diagnosis of AUD, stratify the heterogeneous AUD patient population for clinical trials, select optimal therapy, and monitor treatment response and disease progression. As a first step toward these goals, Dr. Ferguson analyzed gene expression patterns in paired blood and brain samples from mice subjected to chronic intermittent ethanol (CIE) exposure, a mouse model of alcohol dependence. Blood gene expression signatures of CIE predicted a pharmaceutical that reduced alcohol drinking in mice, and predictive models built from blood profiles distinguished between CIE and air-exposed mice with high accuracy. These results lead to the hypothesis that blood can serve as a proxy for brain tissue in molecular-based diagnostic or therapeutic tools and advance personalized medicine approaches for AUD. However, it is not known whether there is a biological signature of AUD in blood from a human population. Furthermore, there is a need for biomarkers to predict and monitor treatment success and it is unknown whether blood gene expression profiles might be useful in this regard. The CIE blood signature used in the previous study assayed the transcriptome only at a single time point. Therefore, the dynamics or ongoing transition of important gene regulatory functions were not investigated. These gaps in knowledge will be addressed in proposed Aims by analyzing blood profiles (1) across multiple time points throughout alcohol withdrawal in humans (Aim 1), (2) across multiple time points through the development of CIE-induced alcohol dependence in mice (Aim 2), and (3) before and after treatment in humans and mice (Aim 3). The overarching hypothesis is that genomic profiles from blood will improve the clinical management of AUD including diagnosis, prognosis, and predicting treatment response. These Aims and the accompanying training plan were designed to build on Dr. Ferguson’s previous research experiences and facilitate new scientific training in clinical alcohol research and biostatistical analyses of longitudinal data, time-to-event outcome data, and treatment effect estimation. The proposed Pathway to Independence Award will generate new knowledge about a relatively unstudied area in alcohol research and enable Dr. Ferguson to establish a solid framework for building a successful research program as an academic translational neuroscientist in the alcohol field.

NIH Research Projects · FY 2025 · 2023-09

Abstract: Spike glycoprotein (S-protein) is one of the viral transmembrane proteins on the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). S- protein plays a crucial role in mediating the initial entry of viral genome into the host cell by binding to the human angiotensin-converting enzyme 2 (ACE2) and then inducing fusion between the virus envelope and cell membrane. Thus, S-protein is a target of choice for diagnostic and therapeutic assays, including neutralizing monoclonal antibodies (nAbs). To date, the conformations of S-protein and its molecular assemblies with ACE2 and/or nAbs have been mainly determined by structural techniques, including crystallographic and electron microscopic methods. These structural studies allow us to understand the molecular basis underlying viral entry and to further develop treatment and preventive therapeutics for COVID-19. However, these resolved structures are rather “static snapshots” compared to the dynamic nature of proteins in physiological conditions. Due to the technical difficulties, our knowledge about the real-time structural dynamics of S-protein and its real-time interactions with host receptors, nAbs, and the other relevant biomolecules, which may have functional significance, is still very limited. In this proposal, my lab will develop a bio-mimicking reconstitution system and apply a cutting-edge structural imaging technique, high-speed atomic force microscopy (HS-AFM), for real-time observations of S- protein’s structural dynamics in close-to-native environments and under various conditions. We will also develop novel methods to quantitatively characterize the architecture of molecular assemblies comprising S-protein, ACE2 receptor, nAbs, host proteases and enzymes, and biological membranes, which can mediate the membrane fusion and viral entry processes. Specifically, we will identify the “real-time” structural dynamics of S- protein in different states and visualize how the state transitions happen, for example, during ACE2 binding, nAbs attachment, and the structural cleavages in S-protein subunits. My lab will further develop correlated fluorescence microscopy and HS-AFM to study these dynamic events associated with S-protein on the mammalian cell surface. The biophysical and biochemical information acquired in our proposed experiments will provide a comprehensive molecular understanding of the conformational states of S-protein, intermolecular interactions between S-protein and binding molecules (ACE2 and nAbs), the conformational changes in S- protein for initiating membrane fusion processes for viral entry, and how the mammalian cell surface impacts the S-protein. The developed methods here can further apply to the other receptor-mediated membrane fusion systems for cell entry.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY / ABSTRACT Bisphenols (BPs) are commonly used plasticizers that are present in many plastics, such as food storage containers and reusable water bottles, as well as lining of food cans. BP exposure via oral consumption is ubiquitous in human populations with inter-individual variation in the levels of exposure. Prenatal exposure to BPs has been associated with negative neurodevelopmental and behavior outcomes for children in human epidemiological studies as well as in rodent models of prenatal BP exposure. BPs such as BPA and other “BPA-free” structural analogs, including BPF and BPS, have been shown to act as endocrine disruptors that primarily affect estrogen receptor signaling. Previous studies demonstrate that BPs can bind to both estrogen receptor alpha (ERα) and estrogen receptor beta to exert effects on gene transcription. Studies using animal models and human placental tissue have demonstrated that BPs can cross the placenta and exert effects directly on the fetus. Maternal BPs may also disrupt estrogen-dependent changes in the maternal brain which impairs postnatal maternal care. Both prenatal BP exposure and altered maternal care are associated with changes in DNA methylation levels across the genome which can lead to stable changes in transcript abundance of affected genes. However, the underlying mechanisms linking prenatal BP exposure and DNA methylation modifications at specific loci are not well-known. In addition, the relative effects of altered postnatal maternal care on offspring with prenatal BP exposure has been understudied. The primary objective of my proposed research is to examine the effects of prenatal BP exposure on DNA methylation modifications at specific loci, notably estrogen responsive elements (EREs), and corresponding changes in gene transcription in the neonatal rat brain. In addition, I will explore a potentially effective intervention, maternal licking-like tactile stimulation, to mitigate the effects of prenatal BP on DNA methylation proximal to EREs in the neonatal rat brain. I hypothesize that DNA methylation changes in response to increased prenatal BP exposure will be enriched in proximity to EREs in the neonatal rat brain and correspond to differences in transcript abundance of estrogen-responsive genes. In addition, I hypothesize that postnatal maternal licking-like stimulation would partially reverse the effects of prenatal BP exposure on DNA methylation modifications proximal to EREs in the neonatal rat brain via increased ERα binding at EREs. Training Potential: In tandem with my proposed research project, I will be developing important research competencies in the developmental origins of health and disease conceptual framework, translational research, and bioinformatic analyses of multi-omics datasets. Following these training experiences, I aim to be competitive for an independent research position and successfully transition to research independence.

NIH Research Projects · FY 2024 · 2023-09

Abstract: Craniofacial differences are among the most common congenital disorders in humans. There are many regulatory factors at play during development of the head and face, with much left to understand. The dysfunction of the ciliogenesis and planar polarity effectors (CPLANE) are a known cause of human Orofaciodigital Disorders (OFD). The Rsg1 subunit of CPLANE is essential for ciliary function, but it remains among the least-studied components, and its mechanisms of action remain largely unexplored. Importantly, my preliminary data suggest that Rsg1 interacts with Fam92, Chibby, and Dzip1, proteins required for basal body docking and transition zone assembly. In this proposed research, the craniofacial developmental differences after disruption of Rsg1 and Fam92 will be explored in a model animal, with a specific focus on neural crest development. In vivo live imaging of ciliated cells will be studied to determine the cellular function of these proteins. Lastly, the biochemical function of the Fam92 BAR domains and disordered regions for membrane morphology will be explored in in vitro systems. Thus, the whole embryo, cellular, and biochemical functions of ciliary proteins Rsg1 and the Fam92- Chibby-Dzip1 module will be elucidated by the proposed work, which in turn will advance our understanding of congenital craniofacial disorders.

NIH Research Projects · FY 2024 · 2023-09

Project Summary The recent convergence of the overdose epidemic and continued racialized police violence has accelerated efforts to mitigate the racially disparate harms of policing toward people who use drugs (PWUD) by aligning law enforcement’s role as first responders with evidence-based public health approaches to drug use rooted in harm reduction. Police departments now commonly equip officers with naloxone, participate in partnerships with healthcare service providers, and have a lead role in celebrated diversion programs targeting PWUD and other marginalized populations. Despite initial evaluations of these approaches showing promise in isolation, widespread and enduring reform across the institution of policing has not materialized. Punitive enforcement of drug offenses remains central to the institution of policing. True harmonization of criminal justice and public health systems requires broad changes to well-established cultural and institutional norms. The proposed R36 application builds on pilot program evaluations and studies examining changes in isolated measures of police enforcement by examining how police officers in Baltimore City, Maryland, negotiate, contest, and make sense of drug policy reforms and guidance from public health authorities in order to understand how the broader cultural shift toward evidence-based approaches to substance use is being translated into policing practices that are ultimately experienced by PWUD. Through in-depth interviews with Baltimore Police Department (BPD) leadership (n=20), observations of BPD public-facing events (n=15), and observational ride-alongs with street-level officers (n=60), this research aims to qualitatively explore, 1) how police leadership integrate harm reduction approaches to drug enforcement into the department’s organizational approach to PWUD, and 2) street-level officers’ beliefs, attitudes, and enforcement practices towards PWUD. Analysis of interview transcripts and observation fieldnotes will be conducted using an abductive approach, drawing on theories and concepts from studies of organizations and organizational change, police culture, risk environments and structural determinants of health. The PI of the proposed R36 application, Bradley Silberzahn, is a doctoral candidate in the Department of Sociology at the University of Texas at Austin (UT Austin). Brad will lead all study procedures, as well as a broader expert panel comprised of faculty from UT Austin, Johns Hopkins Bloomberg School of Public Health, and New York University’s School of Global Public Health. This research will inform ongoing debates over drug policy and the role of law enforcement in harm reduction, as well as identify barriers and avenues for widespread and enduring police reform that facilitates police enforcement practices that are in alignment with, not antithetical to, public health.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Human induced pluripotent stem cells (hiPSCs) have emerged as useful tools in research due to their capacity to differentiate into any adult somatic cell type, including cardiomyocytes (CMs). These hiPSC-CMs have potential use for developing preclinical models of both normal, disease-state, and even patient-specific heart function. However, hiPSC-CM are currently limited in their clinical translatability. One of the primary challenges in deriving cardiac tissue models from hiPSCs is heterogeneity of cardiac differentiation outcomes. Heterogeneity in hiPSC cardiac differentiation results in low, inconsistent CM yield as well as limited control over CM subtype. Overcoming differentiation heterogeneity will improve scalability and lower the cost of utilizing hiPSC-CM based tissue models, as current differentiation methods achieve increased homogeneity by discarding cells that fail to differentiate into CMs or specific CM subtypes, which is inefficient. This proposal hypothesizes that heterogeneity in terminally differentiated hiPSCs arises from heterogeneity between hiPSC clonal lineages that leads to variable response to differentiation cues. Accordingly, accounting for these variable responses should improve homogeneity and consistency of hiPSC cardiac differentiation. To test this theory, a cell barcoding platform, ClonMapper, will be used to address heterogeneity of hiPSC-CM differentiation. ClonMapper uses unique, heritable single-guide RNA (sgRNA) barcode sequences to label cells and track clonal lineage dynamics in response to experimental conditions, such as differentiation cues. This can be used to resolve the transcriptomic heterogeneity of clonal lineages in hiPSCs and connect it to the transcriptomic heterogeneity of those same lineages at different timepoints in cardiac differentiation. Aim 1 of this proposal will verify if ClonMapper is compatible with hiPSC cardiac differentiation by labelling hiPSC populations with sgRNA barcodes and characterizing their pluripotency. Aim 2 will connect transcriptomic heterogeneity of hiPSC lineages to heterogeneity at different timepoints. This will allow identification of which lineages diverge from an hiPSC-V-CM fate and when, characterization of lineages as having high (HDE) or no/low differentiation efficiency (n/LDE), and creation of gene expression signatures associated with HDE or n/LDE lineages. The gene expression signatures of HDE and n/LDE lineages will be used in Aim 3 to identify gene modulators that can be used as added differentiation stimuli to shift n/LDE lineage gene expression to mimicking HDE lineage gene expression, i.e., shift towards an hiPSC-V-CM state. The results from these studies will identify a potential source of heterogeneity in hiPSC cardiac differentiation outcomes, elucidate the underlying mechanisms that cause this, and establish methods for reducing heterogeneity to improve quantity and consistency of specific hiPSC-CM subtype yield (in this case hiPSC-V-CM), advancing the clinical relevance of hiPSC-CMs.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Mitral regurgitation (MR) is a prevalent and deadly disease characterized by the inability of the mitral valve (MV) leaflets to coapt properly, permitting backflow of blood from the left ventricle into the left atrium. The etiologies of MR are complex and diverse, ranging from myxomatous degeneration of the valvular tissue to myocardial infarction to non-ischemic cardiomyopathy. Several types of treatment are available, but the efficacy of these interventions remains suboptimal and unpredictable. One of these options is the relatively recent transcatheter edge-to-edge leaflet clipping technique called the MitraClip procedure (or TEER). Though the procedure is safe, its outcomes in clinical trials, particularly compared to other surgical and therapeutic treatments, have been highly contradictory, largely due to the multifactorial nature of MR. Therefore, it is clear that a predictive approach to treatment selection that accounts for patient specific variations in MV shape and deformation is necessary to optimize long- term patient outcomes. Our group has developed a noninvasive, image-based method for in vivo MV strain estimation which allows us to quantify MV shape and deformation directly from clinically available imaging data. We have also previously demonstrated that this technique can be used to identify predictive, presurgical factors of repair efficacy for ischemic MR patients undergoing undersized ring annuloplasty. However, previous work by our lab and others has focused on limited subsets of MR patients; in order to develop a comprehensive treatment selection guide, simulations must be grounded in a robust understanding of the altered MV biomechanical state in the full range of MR etiologies. Furthermore, the effects of the highly non-physiological focal stress of the MitraClip on the immediate and long-term shape and deformation of the MV leaflets remains almost completely unknown. We will additionally develop a fully predictive finite element simulation of the TEER procedure in order to preoperatively test various MitraClip scenarios directly on a 3D model of the patient's MV apparatus, use our understanding of the MV functional state to predict the 12-month outcomes of each configuration, and select the most optimal option. Therefore, in this study, we aim to (1) establish the pre-operative state of the MV across the MR spectrum and (2) elucidate and predict the consequences of the MitraClip on MV leaflet geometry, behavior, and remodeling in order to explain and ultimately predict the outcomes of this treatment in a patient-specific and quantitative manner.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Natural products (NPs) have historically been a critical source of bioactive molecules, with NPs and their derivatives making up over 50% of FDA-approved small molecule drugs. In recent years, NP-based drug discovery is facing a fundamental barrier in identifying new drugs due to repeated rediscovery of the same or similar compounds, representing limited chemical diversity. Fortunately, since NPs have been evolving over billions of years in trillions of vastly diverse environments, there is an abundance of new bioactive NPs encoded in nature which may be useful as drugs. However, their accessibility is a problem: only less than 10% of NP biosynthetic gene clusters (BGCs) have been connected to existing NPs, leaving the vast majority of BGCs untapped as to what NPs they may produce. The overall goal of this research program is to leverage big-data informatic analysis and biosynthetic investigation to access and convert the tremendous genetic potential of these “orphan BGCs”, BGCs with unknown products, into chemical reality, connecting them to their products and in turn supplying structurally diverse pools of NPs for drug discovery screening. To this end, we propose two research directions: (1) Utilizing our established big-data correlational networking analysis, we have identified hidden proteases missing from the BGCs of almost all class III lanthipeptides. We previously used this method to discover two new families of class III lanthipeptides from Firmicutes for the first time. We will leverage these hidden proteases to further unlock the inherent chemical diversity of lanthipeptides and generate two libraries of natural and non-natural peptides through in vitro enzymatic synthesis and targeted biosynthetic engineering for drug discovery screening. (2) Mining the untapped microbial genetic potential, with an initial emphasis on sulfur- containing NPs and unprecedented biosynthetic pathway hybridization, we have prioritized two promising orphan BGCs with highly unique enzymology and connected them to their native products. The first features a novel S- hydroxylating flavoprotein, potentially involved in the formation of a new sulfur-containing functionality. The second has an unprecedented terpenoid-fatty acid-non-ribosomal peptide hybridization mediated by unusual cross-pathway enzymatic combinations. We will further investigate the new biosynthesis harbored by these BGCs to produce new NPs, inform future genome mining of similar pathways, and enable pathway engineering to further increase NPs chemical diversity. Our significant progress in both research directions supports the feasibility of this proposal as well as our competence to establish a successful and sustainable independent program in this field. We have fostered several key collaborations in bioactivity screening and protein structural biology that further strengthen our research program. In addition, this program will provide opportunities to train undergraduates, graduates, and postdoctoral fellows. Overall, this program is expected to discover new biosynthesis, expand NPs chemical diversity, and facilitate informatics-based NPs discovery and bioengineering to provide promising new drug leads.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Targeting mitochondrial metabolism is an active area of research in Acute Myeloid Leukemia (AML). The cumulative data indicate that AML cells have heightened mitochondrial activity and prefer glutamine as a carbon source compared to noncancerous cells. Nevertheless, a challenge that emerges when trying to target these vulnerabilities one at a time is the continued presence of treatment-refractory AML cells, which ultimately result in relapse or developed resistance. The likely causes are the adaptable nature of metabolic pathways and cell-to-cell variability, either due to local environment or genetics. We propose that loss of mitochondrial nicotinamide adenine dinucleotide (NAD+) will simultaneously block multiple metabolic pathways used by AML with minimal toxicity in healthy cells. We recently published that the SLC25A51 transporter is a critical regulator of mitochondrial NAD+ levels in human cells. SLC25A51 is directly responsible for NAD+ import, and modulation of SLC25A51 expression controls the concentrations of NAD+ in the mitochondrial matrix. Loss of SLC25A51 resulted in depleted NAD+ only in mitochondria and not throughout the whole cell. Until now, there has been no way to selectively deplete mitochondrial NAD+ through an endogenous target. Notably, we have found a broad vulnerability across AML cells to SLC25A51 depletion, including lines that previously were found to escape Complex I inhibition. This proposal will determine the extent that SLC25A51 impacts AML in vivo, elucidate the pathways that this transporter supports, and determine the molecular mechanisms controlling its expression. As there are limited treatment options for patients, the long-term benefit of this work is to establish a rationale for targeting mitochondrial NAD+ through SLC25A51 and to identify additional AML therapeutic approaches.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Broad Impact: Prostate cancer (PCa) is the most commonly diagnosed cancer in American men and in 2022 alone will result in the death of over 34,000 men. PCa mortality is typically caused by disease that has advanced to the metastatic castration-resistant stage (mCRPC) and has spread to distant sites such as the bone, brain, liver, and lymph nodes. Currently, there are no effective or curative therapeutic strategies for mCRPC, which is in part due to high rates of acquired drug resistance to androgen deprivation therapy (ADT) and the standard-of-care chemotherapy drug for mCRPC, docetaxel (DTX). Consequently, there is a critical need for novel and effective therapeutic options for mCRPC. Recent findings have indicated glutamine and related glutamate metabolism as significant drivers of the metabolic reprogramming of mCRPC that contributes to drug resistance mechanisms. Indeed, a metabolic switch has been identified in PCa following ADT that allows the cells to rely on the androgen-independent isoform of glutaminase (GLS1, the enzyme that converts glutamine to glutamate) rather than the isoform that is inhibited by ADT, affording drug resistance. Efforts to chemically inhibit GLS1 to overcome this issue have failed since there is a steady influx of glutamate via the xCT transporter when there are physiologically-relevant levels of cystine. However, our preliminary data shows that concurrently inhibiting GLS1 as well as glutamate dehydrogenase (GDH, the enzyme that converts glutamate to the TCA cycle intermediate alpha-ketoglutarate) may be sufficient to overcome this resistance mechanism across PCa subtypes including mCRPC. The overall goal of this project is to identify novel combinatorial treatments for mCRPC that synergize with DTX to target metabolic vulnerabilities and overcome drug resistance for improved treatment outcomes. Central hypothesis: Concurrent inhibition of GLS1 and GDH in combination with DTX can circumvent drug resistance mechanisms and increase therapeutic efficacy compared to SOC in mCRPC. Aim 1: Determine the metabolic role of GLS1 inhibition with CB-839 plus DTX for the synergistic inhibition of PCa tumor growth in vivo. Aim 2: Elucidate the impact of concurrent GLS1 and GDH inhibition plus DTX on PCa growth, proliferation, metabolism, aggressiveness, and invasiveness compared to SOC in vitro. Aim 3: Evaluate whether combination treatment with a GLS1 inhibitor and a GDH inhibitor plus DTX can synergistically inhibit PCa growth more effectively than SOC in vivo. Experimental techniques including metabolomics, metabolic flux analysis using stable isotope tracers, in vitro and in vivo modeling of mCRPC, and validation of drug treatment efficacy will be undertaken to achieve the research goals. These findings will be used to inform novel treatment strategies to accompany docetaxel to prevent cancer growth, proliferation, and aggressiveness in mCRPC for more effective treatments and improved outcomes for patients with mCRPC.

NIH Research Projects · FY 2025 · 2023-08

Modified Project Summary/Abstract Section The ongoing mental health crisis among Native Americans manifests in extraordinarily high rates mental, emotional, and behavioral health disorders such as anxiety, stress, depression, substance use, and suicide in Native American youth as young as 10. The loss of Tribal Elders and Native speakers has taken an incalculable toll on the cultural ties of language and tradition that flow from Elder Native American generations to the young. Native American people of all ages, have reported some of the highest rates of psychological distress that include depression, anxiety, life stressors, substance use (alcohol, drugs, commercial tobacco use), and suicide. Suicide fatalities for Native American youth between the ages of 15–19 have recently more than doubled. For some Native American communities, suicide is the leading cause of death for youth ages 10- 14. Moreover, Native American youth begin to use alcohol, other substances and commercial tobacco at younger ages (around the age of 11), and at higher rates, than all other group. Thus, interventions to improve Native American youth mental, emotional, and behavioral health outcomes are urgently needed. To meet this urgent need, our overarching objective is to leverage the empirically proven, highly effective, school based, Talking Circle Intervention to promote the mental, emotional, and behavioral health of geographically diverse (rural vs. urban) Native American youth. This study, “Talking Circle for Native American Youth Living Well (A Yo Li)” uses a Community Based Participatory Research (CBPR) approach to evaluate Talking Circle effectiveness, partnering with the American Indians in Texas at the Spanish Colonial Mission (AIT-SCM) in Central Texas, with members living in two geographically diverse areas, rural and urban. “A Yo Li” in tribal languages means “youth”. The Talking Circle Intervention will be implemented as an after-school program in each of the participating schools. After-school programs are an ideal setting for the Talking Circle intervention. After-school programs are preferred by parents, students, and teachers in many Native American communities, as they provide structured, educational opportunities and activities for youth after regular school hours, while parents/guardians are still busy with work or other responsibilities. Based upon the preference of our AIT-SCM tribal partners, and ethical considerations, all students will eventually receive the intervention, thus the control condition is waitlist-control.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Alkylation DNA damage caused by alkylating agents promotes mutations and cancer development. Guanine N7 is targeted by a wide range of alkylating mutagens, carcinogens, and anticancer agents, producing the cationic N7-alkylguanine (N7-alkylG) adducts as major lesions. These lesions have half-lives of several hours to days in DNA and thus can affect DNA replication and transcription. The positively charged N7-alkylG lesions can also undergo further modification to generate secondary lesions such as alkyl-formamidopyrimidine (alkyl-FapyG) adducts. The recognition, repair, and mutagenesis mechanisms of many mutagen/carcinogen-induced N7- alkylG and alkyl-FapyG lesions, except for a few lesions such as N7-aflatoxin B1-G and aflatoxin B1-FapyG adducts, remain poorly characterized, thereby precluding a complete understanding of the contribution of these major lesions to mutations and cancer development. For example, the mutagenic properties of the predominant N7-alkylG adducts produced by the cancer-promoting styrene oxide are unknown. This knowledge gap has been due in part to the technical difficulty in preparing a site-specific N7-alkylG- and alkyl-FapyG-containing DNA, which is ascribed to the rapid depurination of N7-alkylG nucleosides and the facile isomerization of alkyl- FapyG during solid-phase DNA synthesis. To overcome the stability issue of N7-alkylG nucleosides, we have developed a 2’-fluorine technology that prevents spontaneous depurination by increasing the stability of N7- alkylG nucleosides. To solve the isomerization problem of alkyl-FapyG, we have taken a post-synthetic approach that produces alkyl-FapyG-containing DNA from N7-alkylG-containing DNA. Our preliminary studies show that guanine N7 alkylation can influence base-pairing properties by facilitating the formation of the rare enol tautomer, syn base conformation, and/or intercalation. Our central hypothesis is that N7-alkylG and alkyl- FapyG adducts promote mutations and cancer development by altering the base-pairing properties of the damaged guanine. Our long-term research goal is to elucidate the biological impacts of chemically labile alkylation damages and their secondary lesions using innovative approaches such as the 2’-F chemistry, the polβ host-guest-complex system, and post-synthetic DNA modification. The objective is to dissect the biological consequences of N7-alkylG and alkyl-FapyG lesions induced by potent alkylating mutagens and anticancer agents such as nicotine-specific nitrosamine, styrene oxide, nitrogen mustards, and N-methylbenzyl nitrosamine. To accomplish this objective, we will characterize the base-pairing properties and the recognition, mutagenesis, and repair mechanisms of N7-alkylG and alkyl-FapyG adducts using combined tools of synthetic, biochemical, structural biology, and cellular approaches. The successful execution of the proposed programs will greatly advance our knowledge of the impact of carcinogen/drug-induced N7-alkylG and alkyl-FapyG lesions on the base pair conformation, stability, tautomerism, mutagenesis, recognition, and repair, thereby providing important insights into the alkylation damage-induced mutations and cancer development.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Human mesenchymal stem cells (hMSCs) are considered a source for allogeneic therapies to treat diverse diseases. Due to the exponential increase in demand, there is a need for new strategies to produce potent hMSCs to serve diverse patient populations. Currently, conventional planar culture and bioreactors are used as scale-up manufacturing methods. However, these are not specifically tailored for hMSCs expansion. They may alter the cell phenotype and secretome, affecting clinical effectiveness. Further studies to understand the role of substrate mechanics on hMSC expansion are required to achieve reproducible production. Numerous scaffolding alternatives replicate several characteristics of the native extracellular matrix (ECM). However, its dynamic mechanics, which plays a fundamental role in regulating crucial cellular processes, has not been amply studied yet. Furthermore, most in-vitro substrates are static and supraphysiologically stiff. Static substrates have offered a substantial benefit for generating high cell numbers; however, hMSCs have been shown to retain mechanical information, limiting therapeutic capabilities. To address this problem, this proposed research seeks to investigate the role of dynamic cell-matrix interactions and nano-topographical cues on the immunomodulatory potential of hMSCs using a composite of electrospun-fibers encapsulated in a dynamic hydrogel, with the hypothesis that this composite biomaterial will promote high hMSCs production with relevant therapeutic value, while eliminating the limitations reported for the conventional cell culture systems. The K99 period will focus on engineering and characterizing the dynamic nanofibrous hydrogel composites to propel me toward establishing the mechanisms by which they modulate cell quality and potency attributes with relevant therapeutic value (during the R00 phase). In Aim 1, we will develop the dynamic nanofibrous system using a hyaluronic acid hydrogel network crosslinked via dynamic covalent hydrazone bonds that capture the viscoelasticity of ECM in tissues. Four variables, including the encapsulation of the electrospun collagen nanofibers at various densities, fiber diameter, fiber length, and the stress relaxation timescale of the hydrogel will be characterized in this aim to promote hMSC viability and proliferation. In Aim 2, hMSCs cell quality and potency will be assessed by measuring the effect of hydrogel parameters on cellular secretory activity. Immunomodulatory properties will be evaluated by quantifying lymphocyte suppression in co-culture, as well as expression of hMSC surface markers. The capacity of the hMSCs to differentiate will also be assessed. In aim 3, the mechanism linking the biophysical parameters of the nanofibrous hydrogel to hMSC secretory activity will be probed by examining cell adhesive proteins and the activation of transcription factors or sensors of mechanical cues. In sum, the proposed research will lead to new insights to produce hMSCs with high therapeutic value, which will enable new culture substrates that achieve control in reproducibility and cell quality to serve diverse patient populations.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Genome-Wide Association Studies (GWAS) are generated at unprecedented scale in order to link genetic variation to susceptibility to disease and other traits. But as the name suggests, GWAS only generate correlation data-and numerous challenges follow. Combining GWAS data with advanced population genetics tools provides a tremendous opportunity to learn about the genetics and evolution of complex human traits; but we need new statistical methods to realize the possible benefits. My lab will develop such statistical methods and apply them to complex biomedical traits, drawing directly on my previous work and expertise in population genetics, statistical genetics, and computation. First, we will tackle a major concern hindering the adoption of polygenic scores-genetic predictors of complex traits, derived from GWAS: Their poorer performance in groups that differ-whether in genetic ancestry, environmental or social exposures-from the samples in which the GWAS was performed. We will develop a mechanistic understanding of the determinants of the prediction accuracy of polygenic scores, thereby advancing complex trait genetics research for the benefit of all people-in particular historically underserved and underrepresented groups. Second, we will characterize gene-by-environment interactions in complex traits. There is ample evidence that such interactions are common but evidence from GWAS has been underwhelming. We propose a new approach for characterizing gene-by-environment interactions, that might solve this apparent discrepancy: A model that expects concerted changes in magnitude of effects across a large set of variants, e.g. in response to an environmental cue. Where this model fits, it will be germane to phenotypic prediction and to the problem of polygenic score portability across groups. Finally, we will leverage the results of the research described above to study natural selection on complex traits in recent human history. We will focus on two directions: (i) Developing a new method to the study of selection on complex human traits-built upon a marriage between the statistical power of standard GWAS and the immunity of family studies to various GWAS confounders; and (ii) Understanding how variants with genetic effects contingent on the environment evolve. The history of environment-specific selection is hypothesized to have been highly consequential for human health. We will develop new theory and a matching statistical inference tool to understand selective constraint on gene-by- environment interactions, and their consequences for contemporary genetic architecture of complex diseases.