University Of Minnesota

NIH Research Projects · FY 2025 · 2023-08

This Program-project (PPG) application is focused on conducting preclinical studies to understand the molecular basis of immune programming by the rhesus cytomegalovirus vaccine against SIV infection (RhCMV/SIV vaccine), and on defining the role and actions of IL-15 in eliciting vaccine protection against persistent SIV infection. These studies are highly relevant for HIV vaccine development. RhCMV/SIV elicits high frequency and persistent effector-differentiated T cell responses in diverse tissues. Using of this vaccine in the rhesus macaque (RM) model of Simian immunodeficiency Virus (SIV) infection has demonstrated superior efficacy against highly pathogenic SIV challenge over conventional SIV vaccines (overall 59% of RhCMV/SIV vaccinated RM with abrogation of progressive SIV infection). The RhCMV/SIV vaccine response mediates early SIV intercept and replication arrest followed by eventual viral clearance. Vaccine protection is mediated by an MHC-E-restricted immune response of effector memory-differentiated CD8+ T cells unique to CMV-vector vaccination. We have revealed also that interleukin (IL)-15 and the IL-15 response are correlates of protection by RhCMV/SIV vaccination. The IL-15 response is rapidly induced following vaccination, with low prevaccination baseline IL-15 expression/response in blood, followed by rapid induction of IL-15 response networks linked with vaccine protection. This IL-15 dynamic serves as RhCMV/SIV vaccine efficacy outcome predictor. We have defined an inclusive whole blood predictive protective signature (wbPPTS) biomarker of RhCMV/SIV vaccine efficacy, and we will monitor this wbPPTS across RM vaccina cohorts within vivo studies to define the molecular mechanisms of vaccine immune programming. We hypothesize that RhCMV/SIV vaccine and IL-15 together direct expression of specific gene networks across tissues and cells to program effective vaccine immunity and establish the wbPPTS biomarker signature of vaccine protection. To investigate this hypothesis we have designed our PPG with two projects and three service cores including: Project 1. Systemic analysis of the origin and tissue effects of the 68-1 RhCMV/SIV vaccine efficacy-predictive whole blood transcriptomic signature; Project 2: Systems vaccinology of RhCMV/SIV and IL-15 mechanisms of immune programming; Core A, Administration, Core B, Nonhuman primates, and Core C, Systems biology. We feature in vitro/ex vivo and in vivo studies within a Systems Vaccinology design using multiomics 4-platform RNAseq applications (bulk RNAseq, scRNAseq/CITEseq, GeoMx/CosMx, Nanostring) and bioinformatics and statistical modeling approaches to define the systems response across tissues and cell types revealing the molecular basis of immune programming by IL-15 and RhCMV/SIV vaccine.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT: The recent FDA approval of 7T for clinical imaging of the knee, opens the possibility that this ultrahigh field MRI platform will become a more prominent tool in biomedical research and patient management. Musculoskeletal (MSK) research and diagnostic imaging have been traditionally performed at magnetic fields of 1.5 and 3T. While there is increasing availability of ultrahigh field (UHF) 7T instruments and FDA approval of 7T clinical use in the head and knee, only a fraction of the system’s true capability is being exploited. First, approval has only been given for operation in a single transmit mode configuration. As such, clinicians cannot take advantage of the available parallel transmit (pTx) functionality to integrate state-of-the-art solutions for tackling B1+ homogeneity and local SAR management. Even if available, the RF coils and optimization routines to use the pTx functionality do not exist. Second, traditional diagnostic imaging methods for evaluating the knee do not fully exploit the array of morphologic, compositional, and functional data available when performing MRI at UHF. Both the traditional and novel imaging approaches alike benefit from the increased signal-to-noise ratio at 7T. The increased sensitivity can be exploited to provide higher resolution images that are known to have a real and significant impact on diagnostic accuracy in the knee. Furthermore, certain methods are simply too compromised at lower field strengths to be obtained as part of routine imaging at 3T and below including techniques like quantitative sodium imaging and perfusion as measured through arterial spin labeling. In this proposal we focus on the engineering, methodological and protocol developments to realize the full potential of 7T knee imaging and provide a critical translational study focusing on the utility of the methods to impact clinical care. This work will be accomplished by completing four specific aims. Aim 1 will focus on the development, integration, and optimization of a pTx RF coil coil using ultrahigh dielectric constant (uHDC) material and RF management strategies to provide high-resolution morphological knee imaging methods including zero echo time imaging. Aim 2 will implement and optimizequantitative compositional and functional methods for evaluating the knee joint at 7T including T1rho andarterial spin labeling (ASL) perfusion methods. Aim 3 will involve the development and optimization of a sodium (23Na) knee coil using uHDC material and strategies for accurate quantification of 23Na concentration in the articular cartilage of the knee. Finally, the translational Aim 4, will involve a pilot study to explore the ability of the developed technologies and methods to impact patientcare. The technical developments in this proposal will advance knee imaging at 7T and likely accelerate its clinical adoption. The results of the pilot study are expected to have an immediate and important impact,by forming the basis of future longitudinal prospective studies evaluating the effects of medical treatmentson clinical outcome.

NIH Research Projects · FY 2025 · 2023-08

Summary Investigators at the University of Minnesota have teamed up to engineer novel protein activators of the Notch family of cell surface receptors, which are master regulators of T-cell differentiation from induced pluripotent stem cells. This technology will accelerate the development of engineered T cell therapies for treating cancer as well as a range of diseases including auto-immune disorders, infectious disease, immune-deficiencies, graft vs. host disease, and organ transplant rejection. Existing FDA approved engineered T cells are powerful therapeutics yet major challenges remain, including difficulty in differentiating T cells from precursor cell types and difficulty in editing and validating precursor cells prior to differentiation. To overcome these limitations and to enable a transformative jump in T cell engineering approaches, the research team is developing reagents that target and trigger conformational opening of the proteolytic switch NRR domain of Notch1. The Notch NRR buries a cryptic protease site that is normally only exposed to its protease physiologically by tugging forces generated during binding of its ligand on a neighboring cell. The project aims to develop soluble nanobody reagents that functionally pry open the domain and remove the requirement for co-culture with Notch ligands during T cell differentiation. In Aim 1 of the project, phage-display protein engineering is used to identify and optimize nanobodies that selectively bind structurally distinct states of the Notch1 NRR. In Aim 2, these nanobodies are strategically linked together in arrays for creating potent Notch1 activators by inducing conformational “opening” of the NRR. In Aim 3, iPSC cell lines that report Notch1 signaling and T cell commitment will be developed, characterized, and then used in assays monitoring differentiation of iPSCs into T cells under induction by engineered Notch1 activators. In the final stage of the project, the reporter cell lines will be used in benchmarking experiments comparing the performance of Notch1 activators developed in Aim 1 and 2, to industry standard technologies. The milestones of this project are: (a) generating a tool set of molecules that bind selectively to the Notch1 NRR; (b) developing a panel of iPSC reporter systems that monitor commitment and differentiation of iPSCs to T cells that have normal physiologic function including cell killing potential; (c) creating potent and selective Notch1 activators that induce iPSCs to differentiate into T cells with a marked improvement in the efficiency differentiation and expansion over current approaches. This will enable development of improved cancer treatments, and improve health disparities by increasing access to treatment with a standardized iPSC-based cell source that can be frozen and banked for multiple doses while significantly bringing down cost per product.

NIH Research Projects · FY 2025 · 2023-08

During the last 2.5 decades, I have led clinical efforts to develop novel NK cell immunotherapy strategies to treat cancer by advancing lab-based discoveries in the areas of natural killer (NK) cell and IL-15 biology. This work has been supported by a continuously funded and recently renewed NCI P01 (CA111412) grant, now in its 21st year of funding (through 2026). This P01 will serve as the clinical output for the translational work proposed in this R35 application. An R35 award will allow me to further pivot my research program to focus on solid tumors. I started a long-standing NK Cell Program at the University of Minnesota that now includes a team of basic and translational scientists interested in NK cell immunotherapy. My research group has found that exposure to cytomegalovirus (CMV) induces a population of NK cells with potent immune and anti-tumor function that are marked by the expression of the NKG2C activating receptor that recognizes HLA-E, which is overexpressed on many solid tumor cancers. Our highest impact research during the past 5 years is based on an induced pluripotent stem cell (iPSC)-derived NK cell platform designed with attributes of naturally occurring CMV-induced adaptive NK cells. This iPSC platform allows an unlimited number of iPSC gene edits to be performed at the clonal level for mechanistic studies that will be translated into clinical trials. I have used my expertise in NK cell development to help develop methods for directed differentiation of iPSCs to the NK cell lineage (termed iNK) at clinical scale to generate fully functional NK cells for immunotherapy. These iNK cells will be multiplex engineered to enhance tumor-specific activity and persistence in a hostile “cold” tumor environment. My team, who pioneered adoptive transfer of allogeneic NK cells in 2005, has the most experience worldwide, having infused >400 haploidentical NK cell products to treat patients with cancer. We have now made a complete transition away from individual donor blood cell products because of their variability, barriers to gene editing, high cost, and difficulty exporting to the cancer community. The overarching goal of this R35 OIA is to develop novel strategies to specifically target solid tumor malignancies by testing new iPSC edits that facilitate homing and migration, overcome hypoxia, and promote survival after adoptive transfer in patients with solid tumor malignancies. To enhance the specificity and anti-tumoral activity of our iNK cells, we have developed a camelid nanobody specific for B7-H3 that serves as the engager of a novel chimeric antigen receptor (CAR). We have chosen to further study the anti-tumor function of these new CAR iNK cells against two solid tumors (glioblastoma and prostate cancer) that demonstrate oncogenic dependence on the expression of B7-H3. B7-H3 is not expressed at the protein level in normal tissues. We will also compare these CAR iNK cells using the same CAR edited into an iPSC-derived T cell (termed iT). The impact of these investigations is to develop novel off-the- shelf immune cell therapies with potential to change standards of cancer care.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease characterized by chronic wounds and shortened lifespan due to a high incidence of early-onset squamous cell carcinoma (SCC). The highly aggressive nature of SCC in RDEB patients warrants further mechanistic study. High mobility group box 1 (HMGB1) is a serum biomarker of disease severity in RDEB, but its role in tumorigenesis in patients with RDEB has not been thoroughly investigated. HMGB1 is a chromatin-associated protein that functions in the nucleus as a regulator of DNA replication and repair. In response to inflammatory signals, HMGB1 is secreted from the nucleus and functions as a damage associated molecular pattern that stimulates the innate immune response. The central hypothesis of this proposal is that depletion of nuclear HMGB1 in keratinocytes drives carcinogenesis in RDEB by promoting inflammation and accelerating genome instability. This hypothesis will be tested through two specific aims investigating the effects of sequestering HMGB1 in the nucleus. The first aim explores the impact of altered HMGB1 localization on inflammatory response and genomic instability in RDEB keratinocytes. The second aim evaluates the usefulness of small molecule inhibitors of HMGB1 secretion in preventing tumor formation using an in vivo mouse model of RDEB. Successful completion of these aims will provide new information on the biological function of HMGB1 in patients with RDEB SCC. The results of this study have the potential to reveal new drug targets for a fragile patient population in desperate need of safe and effective treatment options. This proposal will be completed at the University of Minnesota under the co-mentorship of Dr. Jakub Tolar, a physician-scientist and pediatric oncologist specializing in RDEB, and Dr. Anja-Katrin Bielinsky, an expert in genome maintenance and DNA repair defects in the initiation of cancer. The complementary expertise of the co- sponsors uniquely positions the candidate to complete the aims described in this proposal and achieve her goal of becoming an academic pediatric hematologist-oncologist leading a research lab focused on examining mechanisms underlying rare genetic diseases and cancer. The career development and fellowship training plans outlined in this application build the foundation for a long and productive career investigating better treatments for pediatric patients with complex and difficult-to-treat genetic conditions.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT This K23 career development project will study the neural underpinnings of impulsivity in adolescent suicidal behavior (SB). The candidate will obtain critical skills and experience in adolescent suicidology and impulsivity research, advanced neurophysiologic and longitudinal methods, and translation of neurophysiologic research to interventional studies needed for a career focused on elucidating brain-behavior mechanisms of adolescent SB. Suicide is the second leading cause of death in adolescence, and rates of adolescent SB are increasing. However, its neurobiology remains poorly understood, and treatments specifically targeting SB are lacking. SB in adolescents is a critical public health problem that demands urgent attention, particularly with research that will rapidly translate knowledge to clinical applications. Negative urgency, a component of impulsivity, is the tendency to act rashly in the context of negative emotion. It has been found to be increased among youth with SB and attempts, and has been linked to impaired inhibition of limbic circuitry by the dorsolateral prefrontal cortex (DLPFC), yet precise mechanisms are unclear. Transcranial magnetic stimulation (TMS) permits noninvasive quantification of DLPFC functions such as cortical inhibition (CI), the process by which cortical interneurons regulate the activity of other circuits. Previous research indicates that adolescents with lifetime SB have reduced CI in the motor cortex that distinguishes them from non-suicidal youth. However, DLPFC CI has not been measured in adolescents with SB, nor is it clear how CI relates to cognitive and emotional systems implicated in SB, such as negative urgency. In order to study CI-related mechanisms of negative urgency in the DLPFC, simultaneous TMS and electroencephalography (TMS-EEG) is required. The candidate proposes a longitudinal study of inhibitory physiology and negative urgency in 40 depressed adolescents with suicidal ideation (but no SB) and 40 depressed adolescents with SB. The study will utilize TMS-EEG and self-report measures of negative urgency to test hypotheses that dysregulated CI is associated with negative urgency, that DLPFC CI is deficient in adolescents with SB, and that CI deficits correlate longitudinally with changes in negative urgency and newly emergent SB. The candidate has prior experience with more basic TMS methods; however, to attain long-term career goals, additional training in EEG analysis, assessment of impulsivity in suicidal adolescents, and longitudinal/neurodevelopmental research methods is necessary. A robust career development plan, with multidisciplinary mentorship and collaboration, will involve intramural and extramural coursework, methodology-specific seminars and training, and a well-defined plan for grant and publication benchmarks. This will ensure the candidate’s successful transition to an independent clinical research career. The long-term goal is to utilize data gathered in this project to design a large-scale longitudinal study assessing neural and behavioral risk factors for developing SB, as well as trials of neuromodulatory treatments that will reduce the transition from suicidal thoughts to behaviors by targeting alterations in CI and negative urgency.

NIH Research Projects · FY 2024 · 2023-08

Abstract Tuberculous meningitis (TBM) disproportionally affects people in low- and middle-income countries and is associated with high morbidity and mortality. Stroke is a common complication in TBM and can lead to severe irreversible neurological disability. Studies on TBM-associated stroke pathology, however, have focused primarily on HIV-uninfected individuals. The overall objective of this proposal is to use transcranial doppler and brain imaging to understand changes in cerebral blood vessels and cerebral blood flow in order to determine changes in cerebrovascular responsiveness, characterize patterns of stroke, and describe long-term neurocognitive outcomes in HIV-associated TBM. The central hypothesis is that in HIV-infected individuals, TBM causes extensive cerebral vascular narrowing and impaired cerebrovascular responsiveness, decreased cerebral blood flow, and ultimately stroke. The central hypothesis will be tested by pursuing three specific aims: 1) describe changes in cerebrovascular responsiveness in HIV-associated TBM; 2) characterize the patterns of stroke in HIV-associated TBM and determine whether it is associated with vasculopathy and/or impairments in cerebrovascular responsiveness; and 3) determine if impaired cerebrovascular responsiveness predicts long- term neurological outcome in HIV-associated TBM. Under Aim 1, transcranial doppler will be used to take repeated measurements of the middle cerebral artery flow velocity and pulsatility index. A subset of study subjects will also have dynamic measurements made in response to stimuli (inhaled CO2, changes in blood pressure, and motor tasks). We will determine whether cerebrovascular responsiveness is impaired in HIV- associated TBM by comparing measurements to those of control patients with no neurological infection. Under Aim 2, all study subjects will have a brain MRI performed within the first 2-weeks of TBM treatment to identify the presence of a cerebral artery stroke and the associated cerebral artery territory. We will compare TCD readings between participants with or without stroke. Under Aim 3, we will examine how TCD measurements at study enrolment relate to the results of neurocognitive testing at month 2 and functional assessment at month 6. Ultimately, the long-term goal of the proposal is to describe how impaired cerebrovascular responsiveness in HIV-associated TBM contributes to the development of strokes and resulting neurocognitive impairment and disability. The research proposed in this application is innovative because it incorporates the use of transcranial doppler at the bedside, to be used in real time, in the care of subjects with TBM. The proposed research is significant because it is expected to provide strong scientific justification for the development of future clinical trials testing targeted interventions to improve cerebrovascular responsiveness and prevent the development of strokes in HIV-associated TBM. Ultimately, such knowledge has the potential of preventing the development of irreversible neurological injury and improving both short and long-term outcomes.

NIH Research Projects · FY 2025 · 2023-08

Abstract This application for an Independent Scientist Award (K02) is designed to advance knowledge important for understanding mechanisms of age-related changes at the NMJ and support the career of Dr. Sarah M. Greising, PhD, an Associate Professor in the School of Kinesiology at the University of Minnesota. As the elderly population of the United States continues to increase there is an ongoing scientific necessity to understand the causes of neuromuscular dysfunction in old age. Understanding neuromuscular dysfunction requires fundamental evaluations of the neuromuscular junction (NMJ) the point of contact between a motor neuron and skeletal muscle fiber. The NMJ initiates muscle contraction and allows physical movement including the ability to breathe across the lifespan. Without innervation, muscle fibers cannot contract, significantly reducing function. The overall objective of this K02 application is to characterize and correct neuromuscular deficiency and low plasticity in the skeletal muscle following both aging and injury by evaluating if shared mechanisms of destabilization of the NMJ exist. This proposal will capitalize on two pathologies that have limited regenerative potential of skeletal muscle by evaluating the NMJ across the aging trajectory of mid- to old-age and following volumetric muscle loss (VML), which is clinically identified as a chronic and irrecoverable loss of skeletal muscle tissue resulting in functional impairments. Both aging and VML injury result in considerable neuromuscular dysfunction, and chronic co-morbidities. My central hypothesis is that progressive NMJ destabilization in aged and injured skeletal muscle create a hostile cellular environment in the muscle that mitigates plasticity and blunts the effectiveness of interventions. I propose two specific aims to address these hypotheses: 1) To understand the limits of diminished innervation and NMJ destabilization; and 2) To determine what spatial changes occur at the NMJ during progressive destabilization. The results of the proposed studies will define cellular mechanisms that contribute to the finite adaptive and regenerative capacity of the remaining muscle after injury and aging and how this relates to NMJ destabilization. The stated goal of the K02 award is to foster the development of outstanding scientists and enable them to expand their potential to make significant contributions to their field of research. This K02 award will advance and reinvigorate Dr. Greising’s scientific development in aging biology by providing protected time to: 1) to build preliminary data and a conceptual framework for a competitive NIA R01-level grant proposal, 2) evaluate mechanisms of NMJ dysregulation in aged and injured skeletal muscle, and 3) build my education and understanding of omics-based tools to develop collaborations able to ask and answer impactful questions linking the physiology of the NMJ (my expertise) and these novel techniques (collaborative expertise).

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Essential tremor (ET) is a common neurologic disorder affecting over 10 million people in the United States. Pathologic synchrony in the cerebello-thalamo-cortical (CTC) network has been considered to underlie the development of ET. Traditional high frequency isochronal deep brain stimulation (T-DBS) in the ventral intermediate nucleus (VIM) of the thalamus has been an effective treatment for ET, however, stimulation related side effects such as dysarthria and ataxia occur in at least 30% of patients. Current spread into unintended brain areas has been considered to underlie most observed side effects related to stimulation. Moreover, habituation, defined as loss of effect despite optimal programming, has been reported in 30-50% of patients. The cause of habituation to DBS is still unclear, however, it may reflect resynchronization in the CTC network. Coordinated reset (CR) stimulation is a novel DBS approach developed to counteract abnormal synchronization in the neuronal network which can address these issues. By using lower current amplitudes and alternating stimulation across multiple contacts of the DBS lead, CR-DBS has been shown in Parkinson’s disease patients to produce acute therapeutic effects comparable to T-DBS as well as carryover benefits that persist for days or weeks after cessation of stimulation. Using lower stimulation current, CR-DBS in the VIM has the potential to minimize side effects from current spread to adjacent structures/pathways and maintain therapeutic efficacy. As a chronic animal model of ET is not available, evaluating the effect of VIM CR-DBS on ET preclinically is not feasible and its effect in patients with ET has never been assessed. The goal of this study is to evaluate the feasibility, safety and efficacy of VIM CR-DBS as a treatment for ET patients, with CR cycle rate and stimulation contacts determined based on the physiological features of tremor related VIM activity. To achieve this goal, we will (1) identify the peak frequency and spatial location of tremor related oscillatory activities in VIM and use these data to guide the selection of CR cycle rate and stimulation contacts, (2) compare the effects of VIM CR-DBS to clinically optimized T-DBS, and (3) characterize the carryover effect of VIM CR-DBS. The findings from this study will provide proof of concept as to the safety and efficacy of CR-DBS as a novel treatment for ET. An understanding of the carryover effect will help determine the dosing schedule of CR-DBS (delivered continuously or intermittently) in future CR studies. If successful the results of this study will significantly advance the development of CR-DBS for the treatment of ET, minimize side effects and potentially reduce habituation, ultimately leading to improved clinical outcomes and a better quality of life for ET patients undergoing VIM DBS.

NIH Research Projects · FY 2025 · 2023-07

In Minnesota, American Indian (34.1%) and non-Hispanic Black (33.4%) women are over twice as likely to be obese during pregnancy than non-Hispanic White women of whom 16.6% are obese during pregnancy. This high incidence reflects the causes of maternal obesity such as low socioeconomic status, high crime rate, and excess exposure to air pollution. Maternal obesity has widespread adverse effects on the offspring including increasing their risk of dying from breast cancer. Maternal obesity also permanently disrupts the mutually beneficial interaction between the offspring and offspring’s gut microbiota, causing gut dysbiosis. Gut dysbiosis in the offspring is characterized by a reduction in the gut bacteria that produce fecal short-chain fatty acids (SCFA). SCFAs play pivotal roles in maintaining healthy immune functions, cellular metabolism, and other critical functions. These compounds act mostly through their receptors GPR43 and GPR41, which are expressed in immune cells and multiple other cell types. Here, we will test the central hypothesis that the composition of commensal gut microbes in the offspring of obese dams is causally responsible for an offspring’s increased susceptibility to mammary tumorigenesis, an effect that likely also reflects altered immunity. We will test this causal link by performing fecal microbiota transfers (FMTs). The role of GPR43 and GPR41 in mediating the impact of maternal obesity on offspring will be tested using CRISPR/Cas9 knockout mice. The potential for clinical translation of our findings will be established by supplementing obese pregnant dams with a commercially available probiotic mix of SCFA-producing gut bacteria and dietary fiber that increases SCFA production. Such a combination has been earlier found to be most effective in reversing loss of critical microbes of healthy gut microbiota from individuals who have consumed an unhealthy Western diet for multiple generations. We will use allografted E0771 and Py230 mammary tumor models and MMTV-PyMT mice developing mammary tumors at about age 3 months. Shotgun metagenome sequencing and mass spectrometry will be applied to study gut microbiota and their metabolites, respectively. Changes in immune cell infiltration and activity will be measured in multiple tissues and compared with the expression of GPR43 and GPR41 in immune cells. Our studies could lead to effective and safe prevention strategies against breast cancer and its growth in the daughters of obese mothers, and be particularly beneficial for communities suffering from health disparities.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Understanding the nature of the molecular interactions involved in human immunodeficiency virus type 1 (HIV- 1) replication continues to provide important insights into the fundamental nature of retrovirus replication. Beyond the importance of such basic science investigations in addressing crucial knowledge gaps in the field, such studies can inform antiretroviral target identification, and have broad applications towards therapy and cure. One of the key aspects of HIV-1 replication that has remained underexplored has been virus particle assembly. The reasons for this have included the challenges associated with the detailed behavior of Gag translocation to the plasma membrane, the engagement of particle assembly sites, the molecular interactions that drive virus particle assembly, and subsequent particle biogenesis and morphology. Integrative, comparative retrovirology has been particularly informative in gaining a deeper understanding of these steps in retroviral replication. For instance, immature particle morphology and the flexibility of the immature Gag lattice differs among retroviruses, which have been insightful for understanding the role of gaps in the Gag lattice and its relationship to relieving stress of lattice curvature. Furthermore, the recruitment pathways for retroviral Gag punctum formation, as well as the role and nature of the actin cortex on assembly site selection and biogenesis also remain poorly understood aspects of the retrovirus assembly pathway. In this application, we propose to investigate comparative analysis of HIV particle morphology and particle biogenesis through innovative state-of-the-art experimental approaches. In particular, we propose to employ cryo-electron microscopy/tomography (cryo-EM/ET), total internal reflection fluorescence (TIRF) microscopy, photoactivated localization microscopy (PALM), dual-color z-scan imaging and double helix-point spread function (DH-PSF) imaging to perform 3D super-resolution imaging and 3D single particle tracking in living cells to investigate 1) high-resolution comparative structural analysis HIV particle morphology and maturation, 2) investigate Gag puncta biogenesis at particle assembly sites, and 3) investigate the role of the actin cortex as a physical barrier in human retrovirus particle assembly. These novel studies harness innovative technologies in order to provide new insights into a highly significant yet poorly understood aspect of the HIV-1 life cycle, address important knowledge gaps in the field, provide insights into potential targets for therapeutic intervention, and inform efforts toward next-generation HIV therapies.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Training of healthcare providers in the reproductive and sexual health (RSH) needs of persons with disabilities (PWD) is critical to addressing a broad array of RSH concerns. Rigorous trials evaluating the effects of curricula on provider behavior are rare. Specifically, we could find no formalized training of healthcare providers in RSH for PWD in sub-Saharan Africa, an environment with the highest rates of human immunodeficiency virus (HIV), sexually transmitted infection (STI), and unintended pregnancy in the world; and where RSH challenges are common and particularly dire for PWD. Reproductive healthcare is a sensitive issue in Africa and PWD are often excluded from consideration. Consequently, a rigorous study of PWD RSH education for healthcare providers is needed if such education is to be widely adopted. Currently, at Muhimbili University of Health and Allied Sciences (MUHAS) in Dar es Salaam, we are conducting a rigorous randomized controlled trial (RCT) of an Afrocentric RSH curriculum training for healthcare providers which was partly adapted from PAHO/WHO for implementation in Tanzania. When we assessed the need for training in RSH needs of PWD in our participants in this trial, nursing, midwifery, and medical students indicated RSH curriculum for PWD to be highly acceptable, needed, and desired by students, and our current NIH-funded RCT has clearly indicated the feasibility of implementation. The next logical step is to create a culturally relevant RSH curriculum to improve student knowledge, attitudes, and skills in providing RSH care for PWD. There are three specific aims. Aim 1 is to conduct a social ecological needs assessment of RSH care delivery for PWD in Tanzania. To determine the most important content when accessing RSH care, we will interview ~20 PWD and ~20 PWD caregivers (total of ~40 interviews) on their experience accessing RSH care. To determine the needs from the perspective of the healthcare workers, we will conduct focus groups of midwifery, nursing, and medical students (3 from each discipline). We will also conduct individual interviews with 12 key informants. In Aim 2, we will further adapt our curriculum, ensure it is culturally tailored to the Tanzanian/sub-Saharan context, and pilot test it. Aim 3 is to evaluate the effectiveness of an Afrocentric, culturally appropriate RSH curriculum for health providers focused on caring for PWD. We will conduct a randomized, controlled, single blinded trial of the curriculum against a waitlist control assessing effects on RSH for PWD knowledge, attitudes, and counseling skills (n=206 students per arm; 412 in total). Hypotheses will test efficacy of the curriculum. We will consult an advisory board of PWD in Tanzania to address structural and cultural issues throughout and use Universal Design (UD)/Universal Design for Learning (UDL) consultant for all study materials. If effective, MUHAS has committed to implement the curriculum for all health students. Given MUHAS is preeminent in health student education across Africa, the curriculum assessed in this study has high potential to be widely adopted as a new standard of training for health professionals across Africa.

NIH Research Projects · FY 2026 · 2023-07

Duchenne muscular dystrophy (DMD) cardiomyopathy is ubiquitous, deadly, and results from mutations in the dystrophin gene. Dystrophin is an essential component of cardiac mechanotransduction (MT) and distributes mechanical stress across the sarcolemma. In DMD, the absence of dystrophin results in cardiomyocytes that are vulnerable to contraction-induced damage, which accelerates disease progression. Our understanding of the early progression of DMD cardiomyopathy is limited due to models that do not fully recapitulate human disease, thus limiting development of effective therapies. Given the essential role of dystrophin in connecting the contractile apparatus to the extracellular matrix (ECM) for MT, it is critical to augment DMD cardiomyopathy models to include cell-ECM engagement with applied physiological force to recapitulate early changes at the organ level necessary to test novel therapies. The human chambered muscle pump (hChaMP) can do just that. The hChaMP is generated by 3D printing human induced pluripotent stem cells and bio-ink to yield a pump that can be pressurized to impose progressive strain. The long-term goal is to determine mechanisms by which DMD cardiomyopathy progresses to develop novel disease specific therapies to prevent cardiomyopathy. The overall objective is to assess the role of altered MT and ECM dynamics in the absence of dystrophin on DMD cardiomyopathy disease progression and to test dystrophin gene editing in a DMD hChaMP with volumetric loading. The central hypothesis is that the loss of dystrophin leads to increased vulnerability to mechanical stress resulting in early altered cardiac MT and ECM dynamics that promote disease progression and that early dystrophin replacement will limit contraction-induced injury by restoring MT and ECM homeostasis and thereby rescue DMD cardiomyopathy. Our central hypothesis will be tested in two specific aims: 1) To evaluate the impact of altered MT on DMD cardiomyopathy disease progression using the hChaMP model system with progressive volumetric loading; 2) To determine the impact of dystrophin restoration with DMD precise gene editing on cardiac remodeling mechanisms dictating disease progression in DMD cardiomyopathy. In aim 1, we will generate a DMD hChaMPs to assess the physiologic impact of increased volumetric pressure on the human DMD phenotype early and later. In aim 2, we will introduce DMD precise gene editing to restore dystrophin both early and late in DMD hChaMPs with volumetric loading and assess the physiologic and transcriptional changes. At the successful completion of the proposed research, the expected outcomes of our study is the characterization of early DMD cardiomyopathy progression with loading and the underlying molecular mechanisms. The proposed research is innovative as it combines enabling technologies to develop a 3D preclinical model of human DMD cardiomyopathy that mimics disease progression and correction with precise gene editing. These findings will have a significant impact on human health by increasing our understanding of disease progression and a strong basis for treating and preventing DMD cardiomyopathy.

NIH Research Projects · FY 2024 · 2023-07

PROJECT ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths and has a dismal 5-year survival rate of 10%. Lethality is attributed to early metastasis, late detection, and therapeutic resistance. Cytotoxic CD8 T cells are the principal mediators of immune surveillance and critical for cancer eradication. However, in PDA and other cancers, CD8 T cells progressively lose their cytotoxic function, and are thus characterized as exhausted. Immune checkpoint blockade aims to correct T cell exhaustion by blocking inhibitory signaling and has revolutionized the treatment of many malignancies. However, PDA is largely resistant. As such, this grant seeks to understand the mechanisms driving CD8 T cell exhaustion in PDA and how PDA evades an activated immune response after immunotherapy treatment. Additionally, this grant will study cancer cell-intrinsic and -extrinsic mechanisms of PDA metastasis, to develop rational therapies. My lab derived new cancer cell lines from immunotherapy resistant tumors, termed escape variants, to study mechanisms of immune evasion. I found that orthotopic implantation of escape variant cells into immunocompetent mice yields multi-organ metastatic disease, which is not seen with the immunotherapy na·1ve cell line. Thus, escape variant tumors can model metastatic disease of PDA patients. Bulk RNA sequencing identified a single gene, Ddr2, that was significantly upregulated across multiple escape variant lines compared to immunotherapy na"ive cells. Ddr2 encodes a receptor tyrosine kinase, is linked to metastasis in other malignancies, and is correlated with reduced patient survival in PDA. Investigation into the tumor microenvironment of escape variant tumors identified an accumulation of intratumoral regulatory T cells (Tregs). I found that Treg depletion significantly reduced tumor burden and metastasis and reduced the development of CD8 T cell exhaustion. I will test the central hypothesis that immunotherapy selects for metastatic cancer cells that promote intratumoral Tregs that cause CD8 T cell dysfunction. In the first Aim, I will investigate how immunotherapy escape drives Treg accumulation and metastasis. First, I will test if Treg accumulation occurs through conventional CD4 T cell differentiation within the tumor. Next, I will test if forced expression of Ddr2 drives metastasis and ablation of Ddr2 prevents metastasis. In the second Aim, I will identify the CD8 T cell subset critical for preventing PDA metastasis. First, I will determine if CD8 T cells are required for the reduction in tumor burden and abrogation of metastasis seen with Treg depletion. Next, I will use single cell RNA sequencing to identify the transcriptional signature of anti-metastatic CD8 T cells enriched with Treg depletion. Completion of the proposed research strategy will reveal mechanisms of metastasis and CD8 T cell dysfunction in PDA and identify a therapeutic target(s) in the hope of reducing PDA lethality.

NIH Research Projects · FY 2025 · 2023-07

CD4 T cells are essential for protection against tuberculosis (TB). But current approaches to TB therapy have not harnessed their potential benefit, and the mechanisms they use to control Mycobacterium tuberculosis (Mtb) have not been completely defined. At the peak of adaptive immunity to TB, millions of Mtb-specific CD4 T cells traffic to the lungs, but very few of them re-encounter their cognate antigen to become activated. The ability to clearly discriminate and isolate these few activated cells from the greater population of inactive CD4 T cells could enable the discovery of important new markers, effector functions, and clonally expanded T cell receptor (TCR) sequences closely associated with protective immunity. To study activated CD4 T cells from Mtb-infected lungs, we used a new approach that combines: (i) a reporter mouse to identify cells actively receiving TCR stimulation in vivo (Nur77-GFP); (ii) BSL3-contained fluorescence cell sorting to isolate them live from tissues; and (iii) single cell RNA sequencing (scRNA-Seq) with CITE-Seq and TCR immune-profiling to interrogate their function and antigen-specificity. In our preliminary work, we discovered that Nur77-GFPHI CD4 T cells express an array of activation markers and costimulatory receptors including OX40. This population was enriched for T regulatory cells and effector T cells with TCR clonotypes localizing to lung parenchyma. Using adoptive transfer, we found that Nur77-GFPHI cells are more protective than their Nur77-GFPLO counterparts. To therapeutically harness activated CD4 T cells in vivo, we treated Mtb-infected mice with a monoclonal antibody that agonizes OX40 and found that this treatment reduced the lung bacterial burden, prolonged survival of infected mice by >100 days and did not cause toxicity. Immunotherapy specifically targeting activated CD4 T cells is a novel approach to TB treatment that has not been thoroughly investigated. In Aim 1 of this proposal, we will define the function of activated CD4 T cells in the lungs of Mtb-infected mice using scRNA-Seq and CITE-Seq. With adoptive transfer and scTCR-Seq, we will uncover how antigen specificity shapes the function and fate of different CD4 T cell TCR clonotypes throughout chronic infection. We will use TCR sequences we generate to develop TCR retrogenic mice that can identify protective Mtb antigens and test the hypothesis that antigen specificity shapes T cell phenotype in TB In Aim 2, we will determine the mechanisms underlying activation marker immunotherapy mediated CD4 T cell control of TB. We will test the hypothesis that OX40 agonist treatment provides protection by selectively modulating the function of both activated T conventional and T regulatory cells. Finally, we will determine how OX40 agonism impacts activated CD4 T cell survival through chronic infection. The studies proposed here represent the first high-definition characterization of the small population of activated CD4 T cells at the site of Mtb infection and the first study exploring the potential of OX40 activation marker targeted immunotherapy in the mouse model of TB. This project will elucidate new mechanisms that may inform the development of more effective TB vaccines and lead to new therapeutic approaches to improve TB treatment.

NIH Research Projects · FY 2025 · 2023-07

Project Summary The global virome is incredibly diverse and emerging viruses threaten global health. Unfortunately, few infection models can recapitulate natural transmission and evolution. Here we propose to bridge natural and experimental systems to study real-time transmission dynamics within and between hosts and infer virus-pathogen relationships. To accomplish this we will leverage a model whereby the natural rodent pathogens from pet store mice are exposed to laboratory mice, rats, hamsters, or deer mice. This model offers a platform for studying acute transmission of viruses between and within hosts via natural mechanisms. One major advantage of this model is that you can access the entire transmission chain from the reservoir tissue, to what is shed, to what either succeeds or fails to replicate in the new host. Aim 1 of this proposal will test the hypothesis that the duration of exposure, interferon and stage of infection in the reservoir shape virus transmission and evolution. In this aim we will address fecal oral and respiratory transmission. Aim 2 of this proposal will test the hypothesis that evolutionary distance and interferon determine the success of cross-species transmission events. To address this we will use wt and STAT2 deficient rats, hamsters, and deer mice. Importantly, this proposal will generate STAT2 deficient rats. Currently, there are no innate immune deficient rats exists and this proposal will provide an invaluable tool for the field. We will measure the capacity of natural mouse viruses to cross the species barrier and determine the impact of evolutionary distance and innate antiviral responses. Overall, this proposal will develop and exploit a model system allows for the analysis of transmission of natural rodent viruses to characterize biological barriers to zoonosis.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY A more complete characterization of auditory cortical processing in humans is critical to understanding auditory perception and cognition. Without it, developing effective treatment options for various auditory processing deficits, such as those rooted in central auditory processing, may not be possible. Currently, there is a lack of consensus regarding how to define and parcellate even the earliest regions of auditory cortex, including primary auditory region A1, highlighting the significant gaps in our overall understanding of sound processing. Traditional approaches to defining primary auditory regions in humans include identifying the macroanatomical landmarks known as the Heshl’s gyri (HG) in each hemisphere and using their locations as a rough approximation of A1. While macroscopic anatomical information, such as the sulcal and gyral patterning in auditory cortex, can provide a rough estimate of where primary auditory regions are located, it is not sufficiently accurate. This is likely due to the high degree of variability in the size, shape, location, and number of HGs found in the auditory cortices of humans. Conversely, attempts to use functional properties—in particular, frequency mapping (tonotopy)—have also been met with limited success, as tonotopic gradients cannot be used to uniquely position the areal boundaries of A1. Aim 1 of the proposed research will exploit recent advances in magnetic resonance imaging (MRI) to non-invasively acquire unprecedentedly high-resolution in vivo human anatomical data at the mesoscopic scale (~0.35mm3), revealing biological information that was not previously available via neuroimaging. Access to this information will allow us to generate detailed, data-driven parcellations of auditory cortices that more closely match the underlying cytoarchitecture. Aim 2 will complement the anatomical approaches in Aim 1 by defining A1 in the same set of individuals, using several high-field cortical and sub- cortical measures of functional activation derived using both task-based and functional connectivity paradigms. The task-based functional data will be used to construct tuning maps for several key perceptually-relevant acoustic features, the parcellation of which will be constrained by the patterns of resting state connectivity between sub-cortical and cortical regions. Work from both aims, which includes mesoscopic MRI, subcortical neuroimaging, computational modeling, and resting state connectivity, will be combined to provide the auditory neuroimaging community with a state-of-the-art multimodal structure-function characterization of primary auditory cortex in humans. To aid in the standardization of auditory cortex characterizations in future studies, this information will be made publicly available, along with an atlas. The long-term goal is a complete characterization and parcellation of auditory cortex in humans. The resulting parcellations in normal-hearing populations will serve as a baseline for characterizing and subsequently developing effective treatments for auditory processing deficits in hearing-impaired populations.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Surgical sites infections (SSIs) are the most common hospital-acquired infection and result in 2 to 11-fold increased mortality, an additional 9.7 days of hospital stay, and annual costs of 3.5 – 10 billion U.S. Dollars. There are myriad techniques implemented to reduce rates of SSIs focusing on reducing skin pathogens. However, if pathogenic bacteria arise from the gastrointestinal (GI) tract rather than skin flora, these preventive interventions may be inadequate to reduce or eliminate SSIs. A lack of knowledge of the source of bacterial pathogens limits our ability to personalize approaches to SSI prevention. There is a growing body of evidence linking the host microbiome to SSI development. Current evidence suggests that skin and GI microbiota diversity may be more predictive of SSI development compared with more traditional SSI predictors. These microbiomes represent a meaningful opportunity to target skin and GI tract dysbiosis as a strategy to prevent SSI. The overarching goal of this research aims to prevent SSIs through interventions targeting the specific source of pathogens and optimizing the host microbial environment. The central hypothesis is that the diversity of the skin and incisional microbiota is predictive of SSI pathogenesis. The hypothesis will be tested through 2 specific aims. 1) First, the study aims to determine if diversity in microbial communities at the incision site is associated with development of SSI by measuring the diversity and microbial community composition of the incisional microbiome at different time points during surgery. Samples will be collected from 300 patients undergoing open GI surgery. A case-control study will be performed comparing alpha diversity using 16S RNA sequencing in 30 patients who develop SSI compared with 30 age-, sex-, diagnosis-, and wound class- matched control patients who do not develop SSI. 2) Next, the study aims to determine if pathogenic strains of bacteria causing SSI are present in the skin or GI microbiota at the time of operation. The strain of bacteria isolated from a subset of 20 patients who develop SSI (from samples collected in Aim 1) will be identified using shotgun metagenomics. Next, shotgun metagenomics will be used to determine whether that specific strain of bacteria was present in the skin and/or GI tract immediately prior to operation. This is innovative and collaborative research assessing the association between SSIs and the microbiome. As SSIs are a global concern, these findings with have global implications and impact. The results from this study will provide important mechanistic details regarding SSI pathogenesis and whether pathogens are present at the skin or GI tract at the time of operation. These results will be used to guide interventional studies targeting modifiable microbiota features that influence SSI development. This study is the first necessary step before performing prospective, interventional clinical trials which can be designed to underscore targeted SSI prevention based on microbial communities.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY: Given the substantial adverse health and economic consequences of diabetes, preventing progression of prediabetes (prevalence of 38% in U.S. adults) to type 2 diabetes (T2D) is a major public health goal. The environment plays a critical role in determining risk for T2D. Urbanization has been associated with increased psychosocial stress and adverse health outcomes. Stress responses cause hormonal changes that lead to insulin resistance, hyperglycemia, inflammation, oxidative stress, and obesity. Increased exposure to air pollution has been associated with higher risk of diabetes and cardiovascular disease. ‘Greenspace’ is defined as publicly accessible areas with predominant naturalistic elements (e.g., trees, grass, water features, etc.). Previous studies in adults support the hypothesis that exposure to Greenspace (‘Green’) may have several health benefits relative to urban built (‘Gray’) environments. These benefits may include better autonomic functioning as assessed by improved heart rate variability (HRV), reductions in anxiety/stress, and enhanced psychological restoration, with Greenspace also serving to buffer air pollution. Walking is the most common form of moderate-intensity physical activity and has a beneficial influence on blood glucose control. Preliminary studies have suggested walking and exposure to Greenspace may act together to improve health outcomes. Yet, aside from our preliminary studies, rigorous experimental studies have not been conducted to examine how regular walking may interact with exposure to urban Greenspace, compared to urban Grayspace, to improve health. These studies are needed to investigate novel and highly generalizable strategies for “Green exercise”, capable of being implemented at the population-level to reduce the burden of disease. Therefore, we will conduct a mechanistic randomized crossover trial to compare differences in physiological, psychological, air pollution, and cardiometabolic risk measures between walking interventions completed in urban Green and Gray environments in adults with prediabetes. This multisite trial will include 180 individuals within metropolitan areas of Minneapolis/St. Paul, MN and Chicago, IL. Walking interventions will be completed in urban Green and urban Gray environments, with participants engaging in 150 minutes/week of walking for 6 weeks in each environment, separated by a 5-week washout. Specific Aims: Aim 1: Measure and compare psychosocial stress between Green and Gray walking. Aim 2: Measure and compare physiological stress over six weeks between Green and Gray walking. Aim 3: Measure real-time individual exposure to ambient particulate matter using personal air pollution monitors with GPS tracking while walking in Green and Gray environments. Aim 4: Measure individual cardiometabolic disease risk scores (index of blood glucose, insulin, lipids, systolic blood pressure, and waist) and inflammatory factors before and after 6 weeks of Green and Gray walking. Aim 5 (exploratory): Examine whether physiological, psychological, and air pollution measures mediate the differential impact of Green vs. Gray walking on cardiometabolic risk and inflammation using regression-based mediation models.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT The goal of this project is to enable the clinical translation of systemically delivered Oncolytic Adenoviruses (OAds) for combined diagnostic imaging and curative therapy of Pancreatic Ductal Adenocarcinoma (PDAC), a devastating disease without effective therapies. PDAC has no effective screening methods, and its spread and metastases are difficult to assess. This new generation of OAds expressing Sodium-Iodide Symporter (OAd5/3- NIS vectors) developed in the Davydova lab, induces uptake of radioactive iodine by pancreatic cancer cells, thus facilitating both SPECT/CT imaging and radiotherapy with 131I. We have demonstrated remarkable pre- clinical data to support the applicability of the OAd5/3-NIS platform to facilitate radioiodine-based imaging and treatment of PDAC. However, the clinical translation of intravenously administrated OAds has been stalled by the lack of an animal model that allows OAd replication. Murine tissues do not support replication of human adenovirus, preventing the use of mice to study biodistribution and off target effects of systemically delivered OAds. Moreover, all rodent systems lack Adenovirus type 3 (Ad3) receptors necessary to bind to Ad3-based vectors (including our OAd5/3-NIS). The need for reliable animal models is critical. Less than 5% of anti-cancer treatments that are promising in murine models are successful in human clinical trials. Therefore, to address this urgent need, we are developing a novel translational swine model of PDAC. Pigs have been attractive as an alternative platform for cancer modeling because of their similarity with humans in terms of anatomy, metabolism, tumorigenesis, genetics, immunity, and body size. Furthermore, as we have recently reported, unlike rodent and canine models, pigs permit replication of both Adenovirus type 5 and Adenovirus type 3 vectors on the level similar to that in humans. Validating systemic administration of OAd5/3-NIS in our swine model of pancreatic cancer is the next step before clinical trials. In this work, we will 1) Produce a novel transgenic KrasG12D/+/TP53R167H/+ swine model of PDAC; 2) Monitor and characterize PDAC tumor development; and 3) Conduct the preclinical studies to evaluate the potential of intravenously administrated OAd5/3-NIS to facilitate both radioiodine-based imaging and radiotherapy with 131I in swine PDAC models. Completion of this proposal will enable clinical translation of systemically injected OAd5/3-NIS vectors for treatment and diagnostic imaging of patients with PDAC, including patients with metastatic cancer. Importantly, these studies will generate essential information on biodistribution, clearance, off target effects, and overall therapeutic potential of other OAds in a clinically relevant, adenovirus replication permissive, immunocompetent model. In addition, our novel pig model will overcome many of the disadvantages inherent in rodents, particularly with respect to size, genetics, cancer biology, metabolism, and immunity leading to translation of safer, more effective cancer treatments for patients with PDAC, a dismal disease with no effective treatments available. 1

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Much progress has been achieved in building computational models that describe how visual stimuli are encoded and transformed across the hierarchy of visual areas in the primate brain. However, relatively little attention has been given to the active component of visual processing, whereby the observer’s engagement in a cognitive or perceptual task can exert substantial influence on stimulus-evoked activity. The long-term goal of the proposed research is to develop models that account for not only stimulus-driven but also task-driven effects in the visual system. To achieve this goal, we invest effort in improving analysis methodology and in generating large high- quality experimental datasets. In Aim 1, we develop and optimize methods that provide robust fMRI measurements at the level of single trials. This includes a method that exploits temporal dynamics to isolate BOLD signals that are more closely related to local neural activity, and an algorithm that improves signal-to- noise ratio in general linear modeling of fMRI data. In Aim 2, we acquire and prepare a high-field (7T) fMRI dataset in which a large variety of tasks are performed on a common set of visual stimuli. This dataset exploits deep sampling of a small number of subjects, and will generate a rich, reusable resource for the fields of cognitive and computational neuroscience. In Aim 3, we exploit the dataset to test an extant model of how top-down influences from the intraparietal sulcus modulate responses in ventral visual cortex and to assess how top-down signals from frontal cortex modulate the fidelity of working memory stimulus encoding in visual cortex. Overall, this multidisciplinary proposal will deliver reusable methods and data resources, and will push computational modeling from the domain of stimulus representation into better understanding how the visual system mediates real-world visual perception and task engagement. Advancing our understanding of the computational mechanisms underlying visual processing in healthy individuals is a critical step for unraveling the nature of sensory disorders such as prosopagnosia and dyslexia.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY/ABSTRACT Medical practice is continuously evolving, with substantial resources allocated to developing medical innovations and enhancing clinical evidence on new and existing treatments. In most cases, new evidence supporting approval by the Food and Drug Administration (FDA) demonstrates efficacy of new treatments; after approval, new evidence for existing treatments may show them to be ineffective or unsafe. Understanding whether and how new clinical evidence is integrated into practice is critical from the perspectives of a) improving patient safety and health outcomes; b) addressing racial and socioeconomic equity in access and use; c) designing programs and policies to encourage use of high value, effective treatments and abandon less effective and harmful treatments; d) containing health care costs by allocating limited health care budgets to their most effective use. The COVID-19 pandemic provides a unique opportunity to study the diffusion of evidence into practice by observing adoption and de-adoption behavior of physicians in response to rapidly changing information about potential treatments and risks of continued use of common drugs in the context of COVID-19. This proposal builds on our prior work on de-adoption of harmful or ineffective medical practices by introducing additional factors that could be associated with the rates and timing of adoption and de-adoption of pharmaceutical treatments. We will examine prescription fills and claims-based use of a targeted set of drugs related to COVID-19 using data from Medicare claims for Fee-for-Service (FFS) enrollees. COVID-19 has particularly affected older adults and the disabled and chronically ill, many of whom face increased risk of severe morbidity and mortality from the disease, and potentially from lack of access to medical care during the pandemic. We will investigate how prescribing of COVID-19-related drugs responds to state-level drug policies, FDA safety communications, and COVID-19 pressures on the healthcare system. We will assess how patient characteristics (e.g., race and ethnicity, age, and key comorbidities), as well as physician and medical practice organization characteristics are associated with these prescribing patterns. Three in-depth COVID-19 case studies will assess uptake and de-adoption of treatments associated with lower quality evidence and more rapid turnaround in a context where public attention is closely attuned to every development and pre-print biomedical manuscripts are discussed at length in the popular press. Exploring adoption and de-adoption of treatments in a context of high uncertainty and high burden and prevalence of disease will provide important insights into the role of information quality in physician decision making. In addition, these cases will allow us to explore the effectiveness of state-level regulation of treatments. In particular, hydroxychloroquine was regulated in some states in response to fears that not enough would be available for people using the drug to treat non-COVID-19 conditions.

NIH Research Projects · FY 2026 · 2023-07

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), is the most common liver disease in the United States and it increases the risk for cirrhosis and hepatocellular carcinoma. Prior research shows that dysregulated lipid partitioning in liver mitochondrial oxidative metabolism pathways [i.e., tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and ketogenesis] is fundamental to liver steatosis and oxidative stress that underlie NAFLD. A traditional view of mitochondrial lipid partitioning is that lipids are fated to ketogenesis when the capacity of terminal oxidation (TCA cycle and OXPHOS) is exceeded, but ketogenesis is limited when the TCA cycle and OXPHOS can support terminal oxidation or under states of high hepatic energy demand. However, this dualist view of lipid partitioning fails to describe heterogeneity in mitochondrial function across the NAFLD spectrum and reveals knowledge gaps in our understanding of how liver mitochondria may coordinate lipid catabolism. The objective of this project is to test the innovate premise that ketogenesis actively supports TCA cycle function and that loss of this salutary coupling contributes to NAFLD. This is based on our preliminary data from mouse models of varying NAFLD severity. Initial studies used phosphatidylethanolamine N-methyltransferase (PEMT)-null mice that exhibit NASH owing to reduced phosphatidylcholine, which is characteristic of human NASH. PEMT-null mice showed lower liver NAD+ and molecular indices of terminal oxidation, however, in vivo TCA cycle flux was unaltered and ketogenesis was increased. This phenotype in mice mimics our preliminary data in humans with NASH which showed elevated ketogenesis and preserved TCA cycle flux. Notably, PEMT-null mice fed a high-fat diet and wild type mice fed a ketogenic diet had lower liver nicotinamide N-methyltransferase (NNMT), which may lead to greater NAD+ salvage to facilitate TCA cycle flux that would otherwise be impaired. In addition, knockdown of ketogenic enzyme 3-hydroxymethylglutaryl-CoA synthase 2 (HMGCS2) in mice on a high-fat diet resulted in lower liver ketogenesis (as expected) and NAD+. Our central hypothesis is that ketogenesis enhances TCA cycle flux via acute and chronic NAD+ provision under conditions of excess lipid availability. This hypothesis will be tested via two Specific Aims. Aim 1 will demonstrate that a ketone body-NNMT-NAD+ axis mitigates liver steatosis by promoting TCA cycle flux in NASH. Mice lacking liver HMGCS2 will receive a Gubra Amylin NASH (GAN) diet and will be independently crossed with liver-specific NNMT knockout mice or provided nicotinamide riboside to increase NAD+. Aim 2 will determine that an increased NAD+/NADH ratio supported by ketogenesis facilitates lipid disposal in response to exercise. HMGCS2 and β-hydroxybutyrate dehydrogenase 1 will be deleted in livers of mice fed a GAN diet to impede ketogenesis and NAD+ provision. Acute and chronic exercise protocols will be completed. Impact: This project will lead progress in creating a paradigm shift by advancing our understanding of how ketogenesis facilitates lipid disposal and informing new therapies for preventing NASH.

NIH Research Projects · FY 2025 · 2023-07

How cells become specified and differentiate at the correct time and place is a fundamental question in developmental biology. Cranial neural crest cells (cNCCs) are an excellent model system to understand this process due to the multipotent nature of the progenitor cells, generally unrestricted developmental potential with known lineage and derivatives, and defined gene regulatory networks. In addition to the gene networks, epigenetic regulators can affect the expression of numerous target genes and may help to explain the differences in penetrance and phenotype between individuals with the same genotype. This is important since defects in neural crest development underlie many human congenital birth defects, such as cleft lip with or without palate and many craniofacial syndromes. Thus, understanding the genetic and epigenetic regulators in cNCC development is key to understanding how cell fate is determined. We hypothesize that PRDM paralogs regulate global gene expression by regulating downstream targets oppositely, including Wnt pathway components, to control the timing of cartilage/bone differentiation within the cNCC lineage. The rationale for the proposed studies is that an in depth understanding of normal cNCC development will provide insights into normal biology and the etiology of neural crest-associated birth defects, many of which are thought to arise from cNCC abnormalities. We will test this hypothesis in the following specific aims: 1) Test the hypothesis that PRDM proteins act upstream of Wnt signaling to control the timing of cNCC differentiation into chondrocytes. We will test the hypothesis PROM paralog activity is required in cNCCs cell autonomously upstream of Wnt signaling to promote differentiation of chondrocytes. 2) Test the hypothesis that Prdm3 and Prdm16 genetically interact to regulate cNCC gene expression and chromatin accessibility. In Aim 2, hypothesis that Prdm3 and Prdm16 genetically interact to control gene expression via regulating transcription and chromatin modification specifically at cNCC and Wnt gene targets. 3) Test the hypothesis that Prdm3 regulates global gene expression by controlling the timing of genomic accessibility of Prdm16. In Aim 3, we will test the hypothesis that loss of Prdm3 leads to global alterations in chromatin state at cNCC progenitor genes via changes in binding of Prdm16 throughout the genome, which controls the liming of cNCC differentiation into chondrocytes. Together, these studies will reveal basic information of how cNCCs differentiate into specific cell types during development. The results of this proposal have the potential to reveal important new insights into cNCC development and how these processes go wrong in disease, with the hope of providing a foundation for the design of therapeutic strategies for neural crest associated birth defects.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Most colorectal cancer (CRC) patients do not respond to immune checkpoint inhibitor therapies due to poor T cell infiltration and an immune suppressive environment. The pathological characteristics of CRC are unique due to the complex microbiome in the tumor microenvironment. Diverse bacterial species and metabolites in the tumor can alter the activity and function of infiltrating immune cells, regulating the tumor's immune environment. Therefore, understanding how tumor-enriched metabolites change the immune environment is crucial for developing novel microbiota/metabolite intervention approaches. We observed that macrophages resembling an M2-like phenotype are in a higher proportion in CRC samples compared to patient-matched normal tissues. Our metabolomic analysis of CRC tissues showed increases in essential amino acids, which strongly correlate with altered macrophage phenotype. Further, monocytes exposed to tumor-enriched metabolites such as tryptophan and phenylalanine increase markers associated with immunosuppressive macrophages. However, whether microbial-derived metabolites alter macrophage polarization and contribute to immunosuppressive phenotypes, which can influence the immune environment in CRC, remains unknown. We hypothesize that altered enrichment of metabolites in CRC correlates with immunosuppressive macrophages and that critical tumor-enriched metabolites modulate the macrophage phenotypes. In Aim 1, we will assess the impact of tumor-enriched metabolites on macrophage phenotype. In Aim 2, we will functionally validate key metabolites influencing macrophage polarization in CRC organoid coculture models. We will use a metabolite screening approach using human primary peripheral blood mononuclear cells from healthy donors and CRC patients to assess whether tumor-enriched metabolites can promote immunosuppressive macrophage phenotypes. In addition, we will use human microsatellite stable and microsatellite instable CRC organoids cocultured with patients matched undifferentiated monocytes and tumor- enriched metabolites to assess shifts in macrophage subtypes. We will identify critical metabolites that can alter macrophage phenotype and function, contributing to a sustained immunosuppressive tumor environment. Impact. Determining interactions between tumor-enriched metabolites and macrophage phenotype will increase the clinical implications of this study by solidifying the premise that the immune environment can be manipulated by endogenous metabolic regulation.