Vanderbilt University

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY Sexual and gender minority (SGM) adolescents are at increased risk for suicide. Approximately 1 in 3 SGM adolescents report having attempted suicide compared to 1 in 10 non-SGM adolescents. SGM adolescents are thought to be vulnerable to suicide because they must negotiate typical stressors of adolescence alongside coming to terms with an SGM identity, which can involve managing exposure to stigma-related stressors including external stressors (e.g., bullying) and navigating taxing internal psychological stressors (e.g., identity concealment). The overarching goal of this proposed K01, entitled Project SPIRiT (Suicide Prediction In Real- Time), is to examine the influence of stigma-related stressors (e.g., bullying, identity concealment) and universal suicide precursors (e.g., hopelessness) on suicidal ideation among SGM adolescents recently hospitalized for acute suicidality. I will utilize state-of-the-art real-time mobile monitoring methods incorporating both ecological momentary assessment (EMA) and global positioning system (GPS) data to pursue the project’s three aims: (Aim 1) develop an EMA+GPS assessment approach capable of capturing both self- reported and place-based exposure to stigma through focus groups with SGM adolescent recent inpatients; (Aim 2) examine precursors to suicidal ideation among SGM adolescent recent inpatients through a 28-day EMA+GPS study; and (Aim 3) develop and refine components of a just-in-time adaptive intervention (JITAI) targeting SGM adolescent suicide risk, informed by qualitative data from focus groups with Aim 2 participants and in-depth interviews with clinicians and JITAI experts. Evidence from Project SPIRiT will lay the groundwork for an NIMH R01 to further test an empirically informed suicide prevention JITAI for high-risk SGM adolescents. To successfully conduct the proposed research project, I will: (1) obtain training in innovative approaches to micro-longitudinal research methodology, including EMA+GPS technology; (2) develop expertise in conducting responsible research with clinical populations and addressing safety concerns; and (3) acquire skills in developing an empirically informed JITAI to reduce suicide risk. By engaging in mentored research with Dr. John Pachankis (primary mentor) and an expert team of co-mentors and collaborators at Yale University and beyond, and participating in coursework, seminars, workshops, scientific conferences, and regular mentorship meetings, I will develop the skills necessary to successfully carry out the proposed research activities and meet my long-term career goal of becoming an independent scientist. Yale University offers rich intellectual and practical resources, an interdisciplinary research and training environment, and professional development and collaborative opportunities to support early-career investigators in launching their independent research careers. The proposed K01 will provide me with the training, mentorship, and protected time necessary to establish an independent program of research focused on SGM disparities and suicide prevention through rigorous scientific inquiry and intervention development.

NIH Research Projects · FY 2024 · 2021-09

Summary Ultrasonic imaging is the most widely used advanced imaging modality in the United States, and excluding planar radiography, it represents 44% of all imaging studies. Unfortunately, ultrasound images are often subopti- mal, and some echocardiography studies show degradation in up to 98% of patients. This rate of degradation and the freehand nature of clinical ultrasound means that image quality is more dependent on operator skill than other advanced modalities, but even with the most skilled practitioners, ultrasound completely fails in 11-64% of clinical tasks. Our overall goal for this application is to fundamentally decrease ultrasound failure rates. Low-quality ultrasound is particularly problematic in echocardiography because of its role as a first-line tool for diagnosing and monitoring cardiac disease. When transthoracic echo is inadequate more costly and time-consuming exams like transesophageal echo or MRI are required. To (1) facilitate the high volumes of transthoracic echo, (2) im- prove patient comfort and safety (i.e. reduce transesophageal echo), (3) reduce cost and procedure time, and (4) maximize physician efficiency, it is crucial to have consistently high quality transthoracic echo. To provide this, we introduce new acquisition and reconstruction solutions for ultrasound imaging. Echocardiographic imaging of the left atrial appendage is growing in importance in lockstep with the rising incidence of atrial fibrillation, which currently effects as many as 2-6 million Americans and is expected to grow to 6-12 million by 2050. In patients with atrial fibrillation, thrombus may appear in as many as 12-26% of left atria with most appearing in the appendage. Therefore, in patients with current atrial fibrillation the left atrial appendage must be screened with echocardiography for thrombus before cardioversion can be performed to return the heart to sinus rhythm. Further, patients receiving a left atrial appendage closure device due to chronic atrial fibrillation require annual transesophageal echo follow ups to monitor the device. For both cases, the gold- standard and standard-of-care is transesophageal echocardiography, and while the left atrial appendage can be visualized transthoracically, is is rarely used because the image quality is considered too unreliable. Ultrasound image quality is degraded by both clutter and thermal noise, so to make clinically transforma- tional improvements to ultrasound–and echocardiography specifically–it is necessary to address both the clutter problem and the noise problem. Addressing both of these issues is becoming increasingly important because recent developments in beamforming have focused on non-linear algorithms (including deep networks), and the performance of these methods are more strongly linked to the noise level compared to conventional beamforming methods. To this end, we introduce both a solution enhancing signal-to-noise ratio using coded excitation and a comprehensive solution for clutter reduction using deep network beamforming.

NIH Research Projects · FY 2025 · 2021-09

1) Background, key gaps in our understanding, and important challenges to be addressed. In response to injury, differentiated adult secretory cells in the gastrointestinal tract undergo metaplasia, or the conversion of one cell type to another. In the pancreas, this is called `acinar to ductal metaplasia' (ADM) and it is thought to function in tissue regeneration. The goals of our laboratory are to generate tools to understand the process of ADM, to identify the consortium of ADM cell types that emerge, and to determine their physiological role in tissue injury. 2) Description of recent progress by the PI. By the end of her post-doctoral work, the PI showed that differentiated, secretory cell types, such as tuft cells, form in the pancreas in response to ADM (DelGiorno et al. Frontiers in Physiology, 2020). Tuft cells are solitary chemosensory cells with myriad roles in mediating inflammation. Combining RNA sequencing, electron microscopy (EM), and mouse models of disease, she identified a functional role for tuft cells, the first description in any model of tumorigenesis (DelGiorno et al. Gastroenterology, 2020). Using single cell RNA sequencing (scRNA-seq) and EM, she has identified the formation of several additional secretory cell populations in ADM of unknown function, which are now being studied in the DelGiorno laboratory. 3) Overview of future research program. We propose to continue our work on ADM by creating a workflow combining Single cell RNA-seq and EM Analysis (SEMA) to study epithelial heterogeneity in pancreatic injury with and without the genetic manipulation of identified cell types. Volumetric electron microscopy (3DEM) will be used as a substrate on which to map the information provided by scRNA-seq to reveal the spatial relationships between cells and infer the function of individual cell types through the study of their organellar content. Our approach leverages scanning electron microscopy (SEM) to conduct a variety of experiments on the same tissue including: [1] imaging of dehydrated whole-mount samples, [2] wide-field “chip mapping” of large areas of ultrathin sections, and [3] large-scale serial section approaches for 3DEM acquisition. Mapping is enhanced using correlative light and electron microscopy to assign molecular markers identified in scRNA-seq by immunofluorescence to cells with defining characteristics in SEM (e.g., nuclear shape, distribution of mitochondria, etc). Our 3DEM datasets are amenable to high-throughput image processing (e.g., segmentation, geometry processing) using machine learning and deep learning tool kits. Together, these tools provide a novel discovery workflow, which will be applied to projects throughout our laboratory aimed at evaluating cellular heterogeneity in ADM. Our goals for the next five years are to use this SEMA approach to build a multi-dimensional atlas of the cellular content and spatial relationships of the various emergent cell types in ADM. Mouse models lacking individual ADM cell subtypes will be used to assess phenotypic effects on cellular makeup, relative positioning, and disease progression. Together, these studies will provide invaluable insight into the role of ADM in tissue healing and regeneration in the pancreas.

NIH Research Projects · FY 2024 · 2021-08

PROJECT SUMMARY/ABSTRACT Hospital acquired infections are a major problem in the United States, affecting approximately 2 million patients and causing at least 90,000 deaths ever year. New strategies are needed to combat these infections, especially in light of rising antimicrobial resistance rates among pathogens. Vaccines, one of the most impactful medical technologies in history, are based on adaptive immunological memory responses, which are long lasting and antigen specific. It is now known that cells of the innate immune system also can mount memory responses, but unlike adaptive memory responses, they provide protection against a broad variety of pathogens. This phenomenon is termed innate immune memory or trained immunity and is a potential solution for preventing infections in vulnerable populations. The mechanisms behind innate immune memory are not well understood. Toll-like receptor 4 ligands, including the vaccine adjuvant monophosphoryl lipid A (MPLA), induce innate immune memory in macrophages. MPLA treatment of macrophages causes metabolic reprogramming as well as increases antimicrobial functions in vitro. In vivo, it protects against Gram-positive bacterial, Gram-negative bacterial, and fungal infections. Our preliminary data shows that MPLA treatment induces high expression of Immunoresponsive gene 1 (Irg1), the enzyme which catalyzes production of itaconate, leading to improved bacterial clearance, an effect that is reduced in Irg1 knockout mice. Itaconate is known to alter metabolism through inhibition of succinate dehydrogenase. It is also an antimicrobial metabolite recently discovered to be delivered to bacteria-containing vacuoles. Based on these findings, we hypothesize that Irg1 and itaconate enable the generation of innate immune memory by facilitating macrophage metabolic reprogramming and augmenting lysosome-mediated antimicrobial functions. Aim 1 will determine the role of Irg1 in generation of the memory phenotype in vitro. Irg1 knockout bone marrow-derived macrophages (BMDM) will be studied to determine the contribution of Irg1 to the metabolic and functional changes associated with memory. Treatment with exogenous itaconate will be explored to determine its ability to induce innate immune memory separately from Irg1 activation. Aim 2 will explore the contributions of Irg1 to MPLA-induced protection against infection and disease tolerance in vivo. Knowledge of the mechanism of innate immune memory is critical to its translation to the clinical setting. This project will be undertaken as part of physician-scientist training through the Vanderbilt MSTP.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Schistosomiasis is endemic in 54 countries and infects nearly 240 million people worldwide per year. Sub- Saharan Africa endures a disproportionate burden, accounting for over 90% of infections. In Kenya, the site of our study, 17 million out of the nation’s 50 million citizens are at risk for schistosomiasis infection. Of the at-risk population, school-aged children represent the primary risk group, and the many morbidities of infection are amplified over a child’s lifespan. After decades of mass drug administration, it is clear that more aggressive and targeted interventions are necessary to move towards the elimination of schistosomiasis. Unfortunately, this recognition has not yet resulted in the development of the tools that public health officials need to make this transition. To address this unmet need, we propose the development of the MEDSCAN (mobile enabled diagnostics for schistosomiasis control analytics) platform to improve schistosomiasis surveillance efforts. This software package builds off of the successful history that we have had in the development of image-processing algorithms for diagnostic purposes. MEDSCAN will consist of a mobile application that can analyze point-of-care diagnostic tests and a web-based administrator dashboard for viewing real-time operational performance metrics and other advanced analytics. We have assembled an interdisciplinary consortium that consists of mobile health software and global health expertise at Vanderbilt University, a world-leader in schistosomiasis diagnostics from Leiden University Medical Center, and a successful history of neglected tropical disease program management and field research at the Kenya Medical Research Institute. To meet our shared goals, our specific aims will: 1) evaluate the cellular network in Western Kenya in preparation for mobile health research, and develop the mobile and web platforms; 2) perform a usability analysis on the platform and make iterative refinements; 3) complete a thorough laboratory evaluation of the MEDSCAN application and a single-site pilot systems check; 4) use the MEDSCAN platform in a 10-month, observational surveillance study. By transforming the already widely utilized point-of-care test into a “connected” diagnostic, MEDSCAN can serve as the gateway to high-resolution surveillance that is necessary to shift efforts from control to elimination.

NIH Research Projects · FY 2025 · 2021-07

Cancer nano-formulations for delivery of small molecule drugs are limited by the ability to target only ~10% of the genome. RNAi molecules can, in theory, be designed against any gene of interest, but siRNA use in clinical oncology faces delivery barriers such as nuclease degradation, rapid renal clearance, poor distribution into tumor tissues, and poor cell membrane penetration. To overcome these challenges, most RNAi therapies focus on synthetic lipo- and poly-plex nano-formulations. Unfortunately, while these technologies typically achieve very high delivery into the liver, high-penetrance siRNA tumor delivery remains elusive. The overarching goal of this project is to develop siRNA chemical modifications that provide potent, safe, tumor-penetrating, and molecularly targeted nano-therapeutics against currently undruggable tumor drivers. The approach builds upon our recently published proof of principle siRNA molecules end-modified through a PEG45 linker with a diacyl lipid (siRNA-EG45<L2), which forms a nano-complex with albumin (alb-NC) in situ following intravenous injection. This albumin “hitchhiking” siRNA-EG45<L2 enhances siRNA pharmacokinetic properties, is very safe, provides natural tumor tropism, and increases tumor delivery level, homogeneity of tumor delivery, and tumor:liver delivery ratio compared to conventional nano-polyplexes formed with in vivo-jetPEI (PEI-NPs). The alb-NCs especially outperformed PEI-NPs for accumulating within challenging patient derived xenograft (PDX) tumors that have reduced access to delivery by the enhanced permeability and retention (EPR) effect. The specific goal of this proposal is to further explore and optimize siRNA chemical modifications for in situ formation of effective alb-NCs. We will benchmark new candidates against conventional nano-formulations in simple (xenograft), immune-competent (allograft) and rigorous (PDX and spontaneous) tumor models. This platform will be validated for silencing of the oncogene myeloid cell leukemia 1 (Mcl-1) to treat triple negative breast cancer (TNBC). Mcl-1 is a vetted target with relevance in a broad range of cancers, supporting its use for proof-of-concept. Furthermore, TNBC is a highly aggressive clinical breast cancer subtype with few treatment options. TNBC patients are currently relegated to chemotherapies, and do not typically benefit from molecularly- targeted therapies. This project is uniquely accessible by our multi-PI interdisciplinary team with bioengineering expertise in intracellular biologic drug delivery nanotechnologies (Duvall), chemical synthesis (Uddin), analysis of noncoding RNA transport on serum components (Vickers), Mcl-1 pathway modulation and analysis (Cook), and cutting edge preclinical models, including PDX, for testing experimental therapies (Brantley-Sieders). Our basic science expertise will be supplemented by consultation with Dr. Ingrid Mayer, a medical oncologist involved in breast cancer clinical trials at Vanderbilt. This group will enable previously inaccessible investigations toward development of more effective, tumor-penetrating, and molecularly-targeted TNBC therapeutics.

NIH Research Projects · FY 2025 · 2021-07

The goal of this application is to understand how the t(8;21), the most frequent chromosomal translocation associated with acute myeloid leukemia (AML), sets the stage for secondary mutations to accumulate and develop into AML. Understanding how the encoded AML1-ETO fusion protein alters epigenetic wiring, is critical to finding less debilitating therapies that yield better outcomes. Both AML1 (RUNX1) and ETO/MTG family members also suffer point mutations in solid tumors, and the ETO family members Mtgr1 (CBFA2T2) and Mtg16 (CBFA2T3) are tumor suppressors in mouse models of intestinal neoplasia, so understanding how ETO contacts histone modifying enzymes has great impact outside of the t(8;21). In our preliminary data, we have used CRISPR/Cas9 technology to modify the 3’ end of the endogenous AML1-ETO with FKBP12F36V- HA or 3XFLAG tags to selectively and rapidly degrade AML1-ETO. We have coupled this system with state-of-the-art genomics such as precision nuclear run-on transcription sequencing (PROseq) and Cut&Run to establish a chemical genetic system to unambiguously define the mechanism of transcriptional control by AML1-ETO. Critically, this allows us to define the earliest, and presumably direct, changes in transcription upon inactivation of the fusion protein. Our preliminary PROseq data demonstrate that enhancers within key hematopoietic regulatory genes such as CEBPA are reactivated within 2 hr of adding dTAG47 to Kasumi-1 cell cultures. These novel reagents allow us to define changes in histone modifications and RNA polymerase dynamics to define the action of AML1-ETO at defined loci and throughout the genome. Moreover, our preliminary data already provide a paradigm shift: even though AML1-ETO bound enhancers have been repressed since the establishment of these cell lines, they were reactivated with a time course that matched the degradation of the fusion protein. Thus, continued expression of AML1-ETO is needed to maintain repression, at many loci, while other loci are more permanently silenced. Finally, we used CRISPR to allow rapid purification of AML1-ETO coupled with MUDPIT and identified a new chromatin modifying complex as potentially mediating AML1-ETO-dependent repression. We hypothesize that AML1-ETO recruits histone modifying enzymes to rewire the epigenetic landscape to suppress CEBPA, PU.1 and GFI1B to impair myeloid differentiation. This sets the stage for secondary epigenetic mutations that reinforce these changes such as inactivation of ASXL1/2. We will directly test this hypothesis by defining the molecular contacts that control AML1-ETO recruitment of repression complexes and use chemical genetics to test if these contacts are required for AML1- ETO-regulated transcription.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY/ABSTRACT Regulatory mechanisms underlying the precise control of gene expression in normal and disease states involve multiprotein complexes such as the highly conserved Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex. Although most of the SAGA subunits have been identified, it remains essentially unknown how their functions are coordinated to precisely regulate gene expression. Thus, the SAGA complex represents an ideal paradigm to explore how multiprotein complexes regulate gene expression, and the overall goal of this project is to provide a precise mechanistic and predictive understanding for the coordination of SAGA subunit function. SAGA subunits are organized into “activity modules”. We will focus on the well-established histone acetyltransferase (HAT), TATA-binding protein (TBP), and histone deubiquitinase (DUB) activity modules in SAGA, which contain the best characterized and evolutionarily conserved SAGA subunits, and are implicated in the regulation of chromatin structure (HAT), transcription initiation (TBP) and RNA export (DUB). Our central hypothesis is that SAGA subunits and modules function together to precisely coordinate different steps in gene expression from chromatin regulation to RNA transcription to RNA export. We will investigate osmotic stress induction of high osmolarity glycerol (Hog1/p38) mitogen-activated protein kinase (MAPK) signaling and gene expression in yeast to study SAGA subunit coordination of gene expression. Importantly, we will use a newly developed detailed and integrated experimental and computational analysis of dynamic single-molecule RNA expression (FISH) in single cells to simultaneously quantify and model each of these steps in gene regulation. Excitingly, our preliminary studies have revealed that the histone acetyltransferase Gcn5p increases the dynamics of chromatin states and stochasticity in gene expression but does not regulate basal transcription, transcription initiation, or RNA degradation. We will determine how the specific HAT module subunits regulate chromatin structure and the kinetics of these processes (Aim 1). We will elucidate how transcription initiation is regulated by unique TBP module subunits (Aim 2). And we will reveal how the specific DUB module subunits differentially regulate RNA export (Aim 3). To accomplish these aims, we propose a rigorous framework of quantitative and dynamic single-cell experiments integrated with sophisticated data analysis and predictive single-cell modeling. This innovative approach will mechanistically dissect gene regulation by the medically relevant and evolutionary conserved multiprotein SAGA complex, providing the first comprehensive analysis of multiprotein gene regulatory complex coordination of gene expression within a single experiment. Furthermore, our studies will provide a blueprint to dissect how other multiprotein complexes regulate gene expression.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Individuals with autism spectrum disorder (ASD) exhibit multiple differences in sensory perception, which have now been recognized as a core feature of the condition. Among these sensory differences, decreased sound tolerance (DST; i.e., an inability to cope with everyday sounds) is particularly salient, with a lifetime prevalence of 50–70% in the ASD population. Despite both first-person accounts and empirical studies indicating that DST is a major source of distress and functional impairment in ASD, little is known about the phenomenology or physiologic underpinnings of this symptom cluster, and no evidence-based treatments for DST in ASD are currently available. Some researchers have suggested that the adverse reactions seen in ASD are manifestations of hyperacusis, reflecting disordered loudness perception. However, others contend that exaggerated emotional responses to specific acoustic stimuli underlie these behaviors, indicating that DST in ASD could be a form of misophonia, a psychiatric disorder characterized by excessive emotional reactions to specific “trigger” sounds. To date, little empirical work has tested these hypotheses against one another, and it remains an open question whether DST in ASD reflects hyperacusis, misophonia, or a combination of the two. Furthermore, it remains unknown whether these symptoms are associated with alterations in the peripheral or central auditory system. The proposed study aims to answer these questions using a two-stage approach. In stage 1, we will construct a novel self-report questionnaire that assesses a wide range of DST symptoms spanning the four theoretical domains of loudness, pain, annoyance (i.e., misophonia), and fear. Available measures of DST typically assess only the loudness (hyperacusis) or annoyance (misophonia) dimensions, failing to address a number of other clinically significant symptoms. Utilizing large online samples of adults with and without ASD, we will refine and psychometrically validate our questionnaire for use as a quantitative measure of transdiagnostic DST symptoms. In stage 2, we will recruit adults with ASD (both with and without DST) and healthy controls from the community, characterizing their auditory function using a battery of psychoacoustic and physiological tests. Auditory perception will be assessed using pure tone audiometry, loudness discomfort level testing, and categorical loudness scaling. Underlying auditory physiology will be assessed from the middle ear to auditory cortex using a combination of tympanometry, acoustic reflex testing, otoacoustic emission suppression, brainstem/cortical auditory event-related potentials, and auditory steady-state responses. Objective auditory measures will be compared between diagnostic groups, and relationships between these measures and subjective DST symptomatology (based on established surveys and the novel self-report developed in stage 1) will be explored across the full stage 2 sample. This project will answer the fundamental question of whether DST in ASD represents a variant of hyperacusis, misophonia, or a combination of the two. Furthermore, by relating DST symptoms to underlying physiology, we will determine whether different dimensions of DST can be separated based on their physiologic correlates. Findings from this study are expected to elucidate the psychological and physiological mechanisms of DST in ASD, improving our understanding of this disabling symptom and guiding the development of targeted interventions for the ASD population.

NIH Research Projects · FY 2025 · 2021-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. T32 GM137793 Vanderbilt CBMS Training Program The Cellular, Biochemical, and Molecular Sciences (CBMS) T32 Training Program at Vanderbilt University provides a unique educational and training experience for Vanderbilt graduate students spanning multiple departments and disciplines in the School of Medicine and the College of Arts and Science. The mission and objectives are to train the next generation of scientific leaders in critical thinking, experimental design, and communication/teamwork skills necessary for productive careers in modern biomedical sciences. Laboratory training and cutting-edge research constitute the core of the program, complemented by structured didactic training in each discipline, as well as ongoing mentoring, training in Responsible Conduct in Research, Rigor and Reproducibility, career counseling, leadership, and communication skills. The CBMS Training Program serves a unique role in interdisciplinary graduate training at Vanderbilt by embracing a large preceptor list that spans 11 different departments and programs. We emphasize broad-based, basic, interdisciplinary research encompassing labs that study a range of topics, complementing other training programs at Vanderbilt that are frequently more discipline-specific. Our program promotes intellectual exchange within the biomedical community at the intra- and inter-laboratory levels. We conduct the only university-wide, weekly journal club providing high level scientific presentations across disparate fields from leading researchers in each area. Trainees also participate in dedicated sessions providing opportunities for oral research presentations, training in responsible conduct in research, rigor and reproducibility, career training, and biosafety. Since no one knows with certainty which areas of modern biomedical science will be required in the coming years, it is essential to provide a breadth of training and interdisciplinary exposure to position the next generation of researchers and leaders so they are well positioned to capitalize on new findings, employ critical thinking skills, and be poised for success in traditional and unforeseen areas.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY More than one third of patients in the intensive care unit (ICU) develop ICU delirium, an acute, fluctuating form of cognitive dysfunction that is associated with increased mortality, and longer ICU lengths of stay and days of mechanical ventilation; and disproportionately increases the risk for long-term cognitive impairment. Disrupted sleep in the ICU is thought to contribute to development of ICU delirium, however the sleep-delirium relationship has not been rigorously investigated. Although ICU patients maintain adequate sleep duration per 24 hours, sleep architecture is severely disrupted. Namely, there is a marked decrease in rapid eye movement (REM) sleep compared to healthy adults. Insufficient REM sleep has been associated with neurodegenerative disease such as dementia, which has similar characteristics to ICU delirium. Less REM sleep has been observed in patients with, compared to without ICU delirium, although the temporality is unclear. To date, few studies have investigated sleep prior to development of ICU delirium, and no studies account for objective baseline sleep. We propose a novel approach to address these critical gaps by leveraging a population of thoracic surgery candidates with a planned post-operative ICU admission to enable baseline sleep evaluation. Research: The K99 phase study will evaluate clinical and demographic predictors of percentage of days with ICU delirium or coma (DoDC) in the acute ICU phase (days 1 up to 14) in a retrospective electronic health record study of thoracic surgery patients (N=4,849) at the University of Pennsylvania Health System (UPHS) from 2015-present. As sleep is not routinely assessed in the ICU, it is not possible to evaluate sleep from medical records. Therefore, we will first identify non-sleep predictors of ICU delirium to then be able to evaluate if sleep indices measured in future studies explains a substantial percentage of the variance in ICU delirium not accounted for by non-sleep predictors. The R00 phase study will evaluate the difference in mean minutes of REM sleep per night for patients with and without ICU delirium in a prospective observational cohort study of thoracic surgery candidates from UPHS (N=148). Sleep will be assessed using wireless electroencephalography at baseline (pre-ICU), and post-operatively in the ICU for 3 days each. In a subset of the R00 cohort (N=20), frequency of awakenings from sleep due to the ICU environment will be assessed. Results from these studies will be used to inform development of future sleep-promoting interventions. Training: To achieve overall career goals, the training plan will build upon the candidate’s background in critical care and sleep in healthy individuals by affording her in-depth training in learning about sleep in clinical and ICU populations, and training in ICU delirium mechanisms and development of predictive models. This training will also include advancing her scientific dissemination skills, as well as development of the skills needed to become a leader in the scientific community. A variety of approaches will be used to achieve these goals including formal coursework, hands-on training, and structured one-on-one mentorship.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT Alzheimer’s disease (AD) is the most common cause of dementia; it is increasingly evident that cerebrovascular mechanisms are critically involved in AD pathology. Cerebrovascular disease frequently co- occurs with AD. One driver of this disease is pathologic fibrin deposition (PFD) in the cerebrovasculature caused by resistance to plasmin-mediated fibrin breakdown. PFD is a risk factor for AD, both non genetic (traumatic brain injury) and genetic (ApoE4), and amyloid beta (Aβ) modifies the fibrin clot, making it resistant to plasmin cleavage and worsening PFD. Thrombin cleaves fibrinogen to fibrin and it also activates platelets by cleaving and activating protease-activated receptors PAR1 and PAR4. We have found that platelet PAR4 is responsible for greater platelet activation, thrombin generation, and procoagulant microparticle formation than platelet PAR1. PAR4 is also expressed on endothelial cells, leukocytes, microglia and neurons, where it is activated by thrombin, leading to activation of microglia, blood brain barrier (BBB) breakdown and microbleeds. Lymphocyte PAR4 is required for homing to sites of inflammation and PAR4 expression is inducible under inflammatory conditions, leading to exaggerated local damage. F2RL3, the gene encoding PAR4, is hypomethylated under inflammatory conditions. Our hypothesis is that PAR4 antagonism may minimize PFD by 1) decreasing thrombin generation and fibrin deposition, and 2) blocking lymphocyte infiltration and local tissue damage. In support of our hypothesis, we have found in preliminary data that PAR4 is overexpressed in the cerebrovasculature of 5XFAD mice with 5 familial AD mutations. In addition, we have shown that in aged humans higher levels of F2RL3 expression in the prefrontal cortex are associated with a faster rate of cognitive decline. As a GPCR, PAR4 is an attractive therapeutic target, and we have made significant progress on generating PAR4 antagonists. However, they do not yet have potent activity against the endogenous tethered ligand in either human or mouse platelets, or the DMPK characteristics to be useful in vivo. In Aim 1, we propose to use a computational approach starting with comparative models of both mouse and human PAR4 to screen an ultra-large library using DOCK and ROSETTA, combined with a make-on-demand small molecule library. Based on the success of the Shoichet and Roth laboratories using this approach to generate pM potency, highly selective compounds for GPCRs, we expect to enrich the scaffold diversity of our SAR. We will iteratively screen compounds in mouse and human platelets, then redo the computational screening to further improve SAR. In Aims 2 and 3, traditional medicinal chemistry will then again iteratively improve the properties of antagonists for potency against the tethered ligand, selectivity and in vivo bioavailability. The best antagonist of mouse PAR4 will be tested in 5XFAD mice to determine if it can slow or block progression of AD symptoms. Completion of these aims will result in in vivo tool compounds for determination if antagonism of PAR4 can arrest or slow the development of AD in this severe animal model of AD.

NIH Research Projects · FY 2025 · 2021-05

Exosomes are small extracellular vesicles (EVs) that are secreted from multivesicular endosomes (MVE) and have been recently recognized to promote cancer metastasis. Exosomes carry bioactive proteins, lipids and nucleic acids and are an important but poorly understood component of the tumor microenvironment. We recently discovered that actin-rich invasive structures called invadopodia are key docking sites for MVE in cancer cells, leading to enhanced exosome secretion. Furthermore, we found that the key invadopodia regulator cortactin enhances MVE docking and exosome secretion. Notably, cortactin is gene amplified and overexpressed in a number of cancers, especially in head and neck squamous cell carcinoma (HNSCC). Furthermore, cortactin overexpression in HNSCC is correlated with decreased patient survival and increased metastasis. Based on these data, we hypothesize that cortactin overexpression drives poor prognosis in HNSCC due to its key role in promoting exosome secretion. Furthermore, we hypothesize that key exosome cargoes synergize with cortactin to promote tumor-induced angiogenesis, lymphangiogenesis, and metastasis. Specifically, we have identified EphB-ephrinB signaling as a key angiogenic axis regulated by HNSCC-secreted exosomes. Thus, we propose that both the number and molecular cargo of exosomes drive aggressive HNSCC behavior in a synergistic manner. We will test these hypotheses and leverage our work to identify potential exosomal blood-and tissue-based biomarkers of regional and distant metastasis.

NIH Research Projects · FY 2026 · 2021-04

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and is associated with significant cognitive decline. In over 40% of TLE cases, seizures are not controlled with current treatment options and systemic anti-epileptic drug administration can have major negative side effects, prompting the need for more effective therapies. However, the cellular and circuit mechanisms underlying TLE are not yet understood due to the inherent challenges of studying chronic spontaneous seizures which typically occur within a relatively short temporal window, often on a timescale of seconds to minutes. Using a recently developed molecular tool, which integrates light and calcium to label active cells within a short temporal window, along with a closed-loop system for seizure detection and light delivery, preliminary results identified a distinct cluster of cells within the hippocampus prominently active during seizures. Additional preliminary work identifies this region as also involved during interictal epileptiform events, suggesting it is a critical control node in the development of seizures. This proposal will employ two different models of TLE, a recently developed focal genetic knockout model and the intrahippocampal kainite model, to dissect the role of this ensemble and 1) Determine its involvement in both interictal and ictal activity in chronically epileptic animals; 2) Determine whether optogenetic inhibition of these cells during seizures can control chronic spontaneous seizures and its associated cognitive comorbidities using a transgenic mouse line that provides access to this distinct population of cells; and 3) Determine whether early intervention in this region can prevent the progression of epilepsy and its associated cognitive comorbidities. The candidate has assembled an Advisory Committee comprised of Gyorgy Buzsaki, Liqun Luo, and Alice Ting to support the acquisition of additional training in closed-loop control of neuronal oscillations, cleared tissue imaging and gene expression analysis, and molecular tool development. In addition, the candidate proposes a personalized plan for career development comprised of additional experience in grant writing, teaching, and scientific management to facilitate success as an independent researcher. The candidate’s long-term goal is to develop a career as an independent neuroscientist utilizing multi-scale investigation at the level of molecules, cells, circuits, and behavior to understand mechanisms of neuronal function and their dysfunction in neurological disorders such as epilepsy. Completion of the proposed study will advance the field by 1) Establishing a previously unknown relationship between interictal and ictal activity; 2) Identifying the therapeutic potential of intervention in a previously unexplored area of the hippocampus to control seizures and associated cognitive deficits; and 3) Identify seizure-specific cellular ensembles for further study. The training period afforded by the K99/R00 award will allow the candidate to develop a powerful set of skills and resources to use in her independent career and the interdisciplinary nature at Stanford provides the ideal environment for the candidate to carry out the research and training plan successfully.

NIH Research Projects · FY 2025 · 2021-04

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of dementia in the United States. Unfortunately, there is no cure for AD. Drug discovery for AD has suffered significant failures, many at late stage clinical trials, partly due to our poor understanding of AD pathology and the lack of disease-relevant and human-relevant discovery and development models. This calls for team efforts with diverse and complementary expertise to tackle the challenges together, by developing innovative approaches from multiple angles to achieve the goal of identifying AD drugs. In this application, we propose three complementary Specific Aims that together aim to identify FDA approved drugs with repurpose potential for AD, from distinct but complementary angles that act synergistically to boost the likelihood of success. AD is a highly heritable disease, with an estimated heritability of 70%, highlighting the critical role of genetics in understanding the disease etiology. Recent genetic studies have identified over 30 loci, enabling us to dissect the genetic architecture of AD, including the biological processes and cell types involved in disease etiology. In particular, we aim to dissect the highly polygenic AD etiology into distinct pathophysiological components to guide drug repurposing, which is only feasible in recent years thanks to large scale GWAS and massive genomics data available publicly (Aim 1). In parallel, we will mine millions of electronic health records (EHRs) to identify drugs that reduce AD risk and cognitive decline, by developing phenotyping algorithms from EHR for AD related phenotypes (Aim 2). In addition, we will develop a high­ throughput screening (HTS) gene expression profiling assay and use human induced pluripotent stem cell (iPSC) models to identify candidate compounds, and will further test the efficacy of the candidates in both patient-derived iPSC lines and AD mouse models (Aim 3). The three aims are complementary and synergistic, in the sense that they independently tackle the same problem from drastically distinct angles, while findings from one can be served as validation for others. Altogether, leveraging distinct and complementary expertise, we expect to yield bona fide repurposable drugs for AD with orthogonal support.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY / ABSTRACT This proposal investigates in the nonhuman primate how attentional load changes the behavioral and neural strategies for flexibly learning object relevance. High attentional load characterizes real-world learning scenarios with multiple, multidimensional objects. Evidence suggests that the neural mechanisms underlying learning during high attentional load fundamentally differs from neural mechanisms used to learn under low load. Our proposal elucidates how learning at increasing attentional load (1) changes the cognitive subcomponent processes used to succeed learning, (2) changes which brain areas are used to flexibly learn, and (3) recruits additional neural circuit mechanisms to realize fast adjustments. First, we will address the specific behavioral subcomponent processes used for learning the relevance of objects in environments with increasing number of visual feature dimensions reflecting increasing attentional load. Simple learning can be achieved efficiently with a hybrid mechanism that uses working memory (WM) of recently rewarded objects to guide future choices together with slower reinforcement learning (RL) for updating longer- term value expectations. When attentional load increases working memory breaks down, and efficient learners flexibly adjust their exploration rates and attentional prioritization to speed up reinforcement learning. Our proposal quantifies these changing learning strategies with multi-component WM-RL modeling. Second, while subjects learn with varying strategies which features to use for making a decision, we will test the causal role of three brain regions implicated to realize the respective learning mechanisms. We use transcranial focused ultrasound stimulation to induce transient, fully reversible lesions allowing to functionally disrupt confined neuronal ensembles. With this tool we elucidate the hypothesized contributions of ventrolateral prefrontal cortex to learning using fast working memory of rewarded objects, the contribution of the anterior cingulate cortex in adjusting exploration strategies and the contribution of the anterior striatum for attentional biasing of slower reinforcement learning of the highest reward-value object within a complex, multidimensional feature space. Third, our project elucidates how the local circuits in each of the three brain areas contribute to successful learning with varying strategies. We use massively parallel recordings of single neuron activity in ventrolateral prefrontal cortex, anterior cingulate cortex, and anterior striatum to extract those cell classes whose firing encodes the key learning variables. We expect that subclasses of interneurons maximally correlate their firing only during those periods when the area specific learning strategy is realized. This approach pinpoints the cell classes that maximally correlate with choice probabilities, prediction errors, working memory, and exploration rates when subjects adjust their learning strategies to successfully learn the relevance of objects with real-world complexity.

NIH Research Projects · FY 2025 · 2021-02

The Initiative for Maximizing Student Development at Vanderbilt University (V-IMSD) seeks to continue advancing diversity and inclusion in our PhD graduate programs. V-IMSD is firmly grounded in a successful 15 year history of the Vanderbilt IMSD in promoting diversity, so that we have become a top producer of PhDs awarded to historically underrepresented students. We know that diversity in all its forms, including a diversity of individuals, thoughts, and approaches, will be essential to adequately prepare the next generation of scientific investigators and realize the potential of biomedical research. And yet we also know that the benefits of diversity are only fully realized through inclusion. Thus, V-IMSD will serve as an essential catalyst for creating a more inclusive training environment. Reflecting the inextricable link between diversity and inclusion, the purpose of this training program is to ensure that students from diverse backgrounds and identities not only acquire the knowledge and skills to pursue successful careers in biomedical research and related areas, but do so in an environment that provides a sense of belonging, where they are comfortable bringing their authentic self to the training experience and empowered to reach their full potential. We plan to continue the IMSD programming that has led to substantial gains in diversity. Our current programming covers five key competencies: research excellence, oral and written communication skills, leadership and management, teaching skills, and responsible conduct of research. Going forward, V-IMSD will expand programming to cover additional competencies such as study design and reproducibility, and quantitative skills. Students will matriculate into V-IMSD as first and second year students, staying associated with the program until degree completion. This enables the development of a community of scholars modeled on Wegner's “community of practice.” Through (1) joint enterprises, such as Data Club, Journal Club and Writing group, (2) mutual engagement, in the form of networking, peer mentoring and social/recreational events, and (3) shared accomplishments via the annual town hall, newsletter, and social media tools, we build a community that offers the mentoring and psychosocial support needed for the success of diverse students. While working to sustain the level of diversity we have achieved in our biomedical PhD programs, we plan to take the training experience to the next level with a focus on improving the inclusivity of our training environment, providing mentee education, or “mentoring up” to help mentees engage with agency in their mentoring relationships, expanding quantitative and computational training, especially for trainees who are not comfortable with these skill sets, leveraging our career development resources in a more UR-centric way, and developing strategic partnerships with the discipline-specific T32 training programs to ensure all students benefit from V-IMSD programming and our leadership efforts on inclusion, particularly focusing on mentorship that is effective and intentionally responsive to different identities.

NIH Research Projects · FY 2026 · 2020-12

Project Summary/Abstract Lung cancer is the most frequent cause of cancer death in the United States among both men and women. If lung nodules can be detected with greater reliability at an early stage, significant improvements in survival rate would be achievable. Chest radiographs are among the most common diagnostic tool used in radiology, and can reveal unexpected incidences of lung cancer. However, even expert radiologists may fail to detect the presence of a subtle low-contrast pulmonary nodule against the high-contrast anatomical background of a chest X-ray, with estimated rates of missed detection of 20-30%. What are the perceptual mechanisms, cognitive mechanisms, and critical learning experiences that determine how well a person can perform this challenging task of lung nodule detection? The PI and Co-Investigator have formed a synergistic collaboration that brings together expertise in human vision, computational modeling and neuroscience (Dr. Tong) in concert with thoracic imaging and biomedical engineering (Dr. Donnelly) to address this longstanding problem with high clinical relevance. This project will develop a validated computational approach for generating a diverse set of visually realistic simulated nodules to achieve the following goals. These are: 1) to characterize radiologist performance on an image-by-image basis in an ecologically valid manner, 2) to develop a novel image- computable model that accounts for expert performance, and 3) to develop a novel learning-based paradigm to characterize the perceptual and cognitive mechanisms of nodule detection, initially in non-expert participants, with the long-term goal of developing a protocol to enhance clinical training. The project will incorporate sophisticated 2D image-based computational methods as well as data from 3D CT segmented nodules to generate a diverse set of simulated nodule examples, each placed in a unique chest X-ray. Success will be evaluated by the following outcome measures. First, radiologists should find it very difficult to tell apart real from simulated nodules. Moreover, their performance accuracy at detecting/localizing simulated nodules should be predictive of their accuracy for real nodules. Second, if the simulated nodules suitably capture the variations of real nodule appearance, then non-expert participants who receive multiple sessions of training with simulated nodules should show improved performance for both simulated and real nodules. This learning- based paradigm will allow for characterization of the perceptual, cognitive, and learning-based factors that govern nodule detection performance. Third, development and refinement of this learning-based paradigm should have the potential to improve nodule detection performance in radiology residents. Finally, the behavioral data gathered from radiologists and other top-performing participants will be used to develop an image-computable model of nodule detection performance. As a whole, this project will lead to a more rigorous understanding of the perceptual and cognitive bases of lung nodule detection, and spur the development of a new learning-based protocol to enhance the training of radiology residents and other medical professionals.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY – Overall. This application proposes the extension of our Tissue Mapping Center (TMC) for the human eye and pancreas within the Human BioMolecular Atlas Program (HuBMAP). The mission of our TMC is to build a platform of integrated technologies for imaging and molecular analysis that enables the construction of comprehensive 3-dimensional molecular atlases of human eye and pancreas. This TMC leverages the unique resources of the Mass Spectrometry Research Center at Vanderbilt University, the world-class clinical environment of the Vanderbilt University Medical Center, the distinguished expertise of Organ Specific Project team leaders in their respective fields and their vast experience in organ procurement and management, and the advanced biocomputational infrastructure available to the TMC through the Data Analysis Core laboratories at Vanderbilt University and the Delft University of Technology, Netherlands to create a capability to molecularly characterize human tissues in 3-dimensions at a level of understanding unrivaled by current technologies. The innovative aspects of the proposed TMC are (1) the integration of imaging mass spectrometry, co-detection by indexing (CODEX), stained microscopy, and autofluorescence microscopy into 3-dimensional molecular atlases, (2) whole organ imaging of the eye, and (3) multi- organ atlases from the same donors through a proposed collaboration with other currently funded TMCs. The application of our platform to organ-specific projects in pancreas and eye will provide a new paradigm of understanding the normal state of these organs across vast scales, both molecular (e.g., lipids, metabolites, and proteins) and spatial (e.g., whole organs to single cells). Furthermore, the involvement of these organs in diabetes, along with organs currently funded by HuBMAP such as kidney and vasculature, will lay an important foundation for future studies striving to understand the progression of diabetes. As a HuBMAP participant, the molecular atlases produced by this TMC will be disseminated to collaborators to generate new hypotheses regarding the function of these important organ systems, enabling new insight into human health and disease.

NIH Research Projects · FY 2024 · 2020-09

Project Summary In this new application we propose a unique training experience that builds upon Vanderbilt University's (VU) extraordinary record of research, training, and fostering diversity in biomedical science and that seeks to create a new paradigm promoting the pathway to biomedical science careers and aspirational life choices for a cadre of underrepresented, postbaccalaureate scholars. This program provides an intellectually, socially and culturally rewarding experience to engage scholars at multiple levels throughout a 12 month training program. We will leverage VU's training expertise to cultivate knowledge and research skills and provide an integrated training program that supports resilience by fostering connections among program participants and their research mentors, peers, and respective communities. A strong multiple-PI leadership team is composed of senior individuals with overlapping expertise in scientific research, mentoring, inclusion, skill development, trainee engagement, and fostering a community of diverse scholars. This team is supported by internal and external board members with demonstrated commitment to training and diversity. We will provide a research intensive immersion for carefully-selected scholars each year. Our pool of distinguished mentors have a diversity of research interests that are aligned with the mission of NIGMS. In addition to an intense, mentored research experience, we will offer unique perspectives and resources, provide a productive framework to fully understand the promise and process of scientific discovery, and foster invaluable connections among scholars and their mentors and communities. We posit that scholars, most especially those from underrepresented groups, must also be trained in how to relate their work to the larger community. They must be aware and knowledgeable of the forces shaping the public's perception of science and how to shape the scientific narrative in both underrepresented and majority communities. Our overall goal is ambitious and innovative; we seek to create scholars that will become the integrators needed to connect science with communities through their academic credentials, ability to build and retain trust, and cultural competence skills. We will fulfill this vision through: an intensive research and training immersion experience, with mentoring as a central component; regular opportunities to interact with their peers and acquire the core values of collaboration, teamwork and personal enrichment; and exposure to the public awareness of science, education, policy and social change. Together, we will thus provide an exciting and comprehensive experience to minority scholars poised to make career-defining, life- changing decisions with future implications for the scientific enterprise across the nation.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Metabotropic glutamate receptor 7 (mGlu7) regulates presynaptic neurotransmitter release widely throughout the CNS and is required for the induction of long-term potentiation (LTP) in the hippocampus and amygdala, suggesting a central role in synaptic plasticity. Recently, primary mutations in the GRM7 gene have been linked to intellectual disability, stereotypies, and seizures, symptoms common in neurodevelopmental disorders. Additionally, we have found that mGlu7 protein levels are significantly reduced in the brains of patients clinically diagnosed with the neurodevelopmental disorder, Rett syndrome (RTT). Loss-of-function mutations in Methyl CpG Binding Protein 2 (MECP2), an epigenetic regulation of transcription, are the major cause of RTT, which is a disease resulting in the development of stereotyped behaviors, motor delays, anxiety, cognitive deficits, autistic features, seizures, and apneas. Consistent with reductions in mGlu7 in RTT patients and model mice, potentiating mGlu7 activity with small molecule positive allosteric modulators (PAMs) corrects multiple synaptic plasticity, cognitive, social, and respiratory phenotypes in mice with an Mecp2 knockout (KO) allele. These early studies employed a compound that was not selective for mGlu7 versus several other metabotropic glutamate receptors. We have recently optimized VU6027459, a highly selectivity mGlu7 tool that exhibits suitable pharmacokinetic parameters for in vivo rodent use. Using VU6027459, we have further validated a role for mGlu7 potentiation in reversing abnormal RTT phenotypes. We propose to use the monogenetic disorder of RTT, in tandem with a drug development campaign, as a clinical entry path for the development of mGlu7 PAMs; it is anticipated that future studies could then expand into alternate indications, such as primary epilepsies or schizophrenia. Our previous RTT studies have focused on patients and mice with an MECP2/Mecp2 allele that is a functional null. A large proportion of RTT patients, however, have single point mutations in the MECP2 gene, and our preliminary data suggest that there are differences in mGlu7 expression levels in human RTT tissue that correlate with specific mutations. This indicates that therapeutics for efficacy testing in this disease require preclinical validation in various mouse models that reflect this clinical heterogeneity. Using an expanded clinical sample set and mice modeling different mutations, we will test the hypothesis that mGlu7 levels may be differentially impacted in the context of distinct MECP2 mutations and determine if specific mutations underlie disease states most likely to exhibit efficacious and safe responses to mGlu7 PAMs or if mGlu7 PAMs will have utility across the entire mutation spectrum. Finally, we will perform biomarker studies that build upon our findings that Mecp2- deficient mice exhibit EEG spectral changes and reductions in REM sleep across the disease course. These findings mirror sleep abnormalities seen in RTT patients, suggesting that EEG represents a clinically translatable and noninvasive biomarker for the program.

NIH Research Projects · FY 2024 · 2020-09

Project Summary The ongoing COVID-19/SARS-CoV-2 pandemic highlights the need for simple, rapid, and cost-effective testing for respiratory infections at the point-of-care, including physician’s offices, urgent-care settings, ambulatory procedural centers, and low-resource environments. The need is particularly notable for respiratory infections, such as COVID-19 and influenza, which can present with similar symptoms yet require distinct management strategies. With its high sensitivity and specificity, RT-PCR is the gold standard for the molecular diagnosis and differentiation among respiratory pathogens. Traditional RT-PCR workflow requires significant control over specimen contents and reaction conditions, with current methods requiring nucleic acid extraction prior to amplification and detection. The net result is increased complexity, cost, and/or turnaround time for diagnosis. In this context, we have observed in recent influenza studies that outstanding analytic performance characteristics can be achieved without RNA extraction, by applying our novel workflow and Adaptive PCR technology. Unlike traditional RT-PCR, Adaptive RT-PCR incorporates mirror-image L-DNA enantiomers— identical in sequence to PCR primers and targets—that modify cycling conditions to match the biochemical sample contents, thus eliminating the need to monitor reaction temperature. The direct monitoring of reaction conditions overcomes many of the limitations of traditional PCR, facilitating direct amplification within the original specimen matrix, simplifying instrument design, and enabling single-tube analyses. SARS-CoV-2 and influenza are both enveloped RNA viruses, with specimens collected in the same manner (i.e. nasopharyngeal swab) and using the same viral transport medium. Therefore, we hypothesize that we may eliminate RNA extraction for this virus, like we have done for influenza, by performing Adaptive RT-PCR directly on clinical specimens. We propose to enable a simplified methodology through Adaptive RT-PCR, creating diagnostics for COVID-19 and other respiratory pathogens without RNA extraction. As a collaboration between biomedical engineers and a COVID-19 diagnostic laboratory, we seek to develop a workflow and instrument that are simple-to-use, cost-effective, and suitable for point-of-care settings, tools that can rapidly inform treatment and management strategies. To achieve this goal, Aim 1 will evaluate the performance of RT- PCR directly – that is, without RNA extraction – using both traditional and Adaptive RT-PCR instrumentation. Aim 2 will develop multiplexed amplification reagents to create a sensitive and specific respiratory panel that detects SARS-CoV-2, four other viruses, two bacteria, and one control target. Ultimately, Aim 3 will design and fabricate a self-contained Adaptive RT-PCR instrument suitable for point-of-care settings, while validating this system using characterized human specimens in a CLIA-accredited lab environment. Completion of this project will result in a novel point-of-care tool for both the established and emerging respiratory infections that threaten public health, facilitating rapid treatment, follow-up, infection prevention, and epidemiologic containment.

NIH Research Projects · FY 2025 · 2020-08

PROJECT SUMMARY Immune checkpoint blockade (ICB) is an immunotherapy that is revolutionizing cancer treatment, but is effective in a minority of patients. Across many cancer types, this can largely be ascribed to an insufficient number or function of tumor infiltrating T cells positioned for reactivation by ICB antibodies. Therefore, there is a critical need for strategies to increase tumor immunogenicity that results in a greater number of patients that benefit from immunotherapy. Our long-term research goal is to improve responses to immunotherapy through the molecular engineering of materials that harness endogenous mechanisms of antitumor innate immunity. To that end, we have developed STING-activating nanoparticles (STING-NPs) – a new class of endosome- destabilizing polymer vesicles (polymersomes) that enhance the cytosolic delivery of cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway. CDNs have poor drug-like properties and therefore suffer from poor cellular targeting, rapid clearance, and inefficient transport to the cytosol where STING is localized. This has restricted clinical evaluation of CDNs to local, intratumoral administration, which is not feasible for many cancer patients with advanced disseminated disease. STING-NPs enhance the potency of CDNs by several orders of magnitude, resulting in increased tumor immunogenicity, inhibition of tumor growth, and improved response to ICB. Our objective in this R01 application is to further expand the utility and therapeutic window of STING-NPs by 1) optimizing their properties for safe and effective systemic administration via an intravenous route, and 2) designing new combination therapies that leverage their immunopharmacological properties to improve immunotherapy responses in melanoma models that are resistant to ICB. We will accomplish this through the following Specific Aims. First, we will re-engineer the polymersome corona to optimize the pharmacokinetics and biodistribution profile of intravenously administered STING-NP to achieve maximal CDN delivery and STING activation in the tumor microenvironment. Second, we will synthesize a new class of modified CDNs that are structurally optimized for increased incorporation and retention into STING-NPs, and will investigate the effect of CDN structure, loading, and stability on immunostimulatory activity and therapeutic efficacy. Third, we will develop rationally designed and clinically relevant chemo- and immunotherapy combinations that target mechanisms of resistance to STING agonists that we have recently identified. Overall, these studies will advance STING-NPs as a platform for increasing tumor immunogenicity and improving outcomes of immunotherapy. In doing so, these investigations will also advance our understanding of relationships between nanocarrier properties, pharmacological behavior, antitumor immunity, therapeutic activity, and toxicity with potential to inform design criteria that are broadly applicable to STING and other innate immune agonists.

NIH Research Projects · FY 2024 · 2020-08

How does our ability to detect motion develop and to what extent does sensory experience contribute? In mice, the detection of motion begins in the retina. There, direction selective ganglion cells (DSGCs) fire more action potentials in response to visual stimuli moving in one direction, called the preferred direction, than visual stimuli moving in the opposite direction, called the null direction. In the adult retina, the preferred directions of DSGCs cluster along 4 directions. The relative orientation of these clusters varies across the surface of the retina, creating a direction selectivity map. The factors which underlie the development of the direction selectivity map is unknown. Two recent experiment indicate visual experience is involved. The first is that the preferred direction of DSGCs cluster along the axes defined by optic flow, i.e. the trajectories of apparent motion mapped onto the retinal surface. The second is from the Feller lab showing that the clustering of DSGCs preferred directions along the 4 axes requires visual experience. In Aim1, I present strong preliminary evidence that demonstrates that the map is established at eye-opening and is not altered after visual deprivation, contradicting a role for activity. Although visual experience does not seem necessary for establishing the direction selectivity map, it may play a role in refining it. For example, raising a mouse in an artificial environment that enriches for specific orientations leads to an overrepresentation of cortical cells that prefer that orientation and the process is thought to be instructive rather than permissive. Therefore, in specific aim 2, I will determine whether altered visual experience, specifically enriched for optic flow, refines the direction selectivity map. Finally, in Aim 3, we explore the factors that influence the organization of the direction selectivity map at eye opening. First, we test whether retinal waves, which are spontaneous depolarizations of retinal ganglion cells that occur exclusively during the first two weeks of postnatal life, play a role. These waves are the primary driver of activity before eye opening in the direction selectivity circuit and even exhibit a propagation bias toward forward optic flow. We propose a pharmacogenetic experiments to determine whether disruption of retinal waves alters the organization of the map. Second, we begin to explore the role of transient molecular gradients that are expressed in the retina when the direction selectivity circuit is developing. Specifically, I provide evidence that the EphB receptor of the ephrinB axon guidance molecule exhibits a similar distribution gradient as the dorsal- ventral DSGC preferred directions. We will then look at maps in a mouse lacking EphB receptors in the retina.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY/ABSTRACT Pleiotropy, where one gene affects multiple discrete traits, presents an interesting puzzle for evolutionary biologists because mutations that are adaptive for one trait could antagonize the function of another. Recent studies in humans and other organisms have suggested that pleiotropy is extremely common, raising fundamental questions about the function and maintenance of pleiotropy over evolutionary time. There is a long-acknowledged but poorly studied observation that many innate immune signaling pathways in plants, insects, and other taxa also play dual roles in modulating development and other conserved traits. Several studies on genomic signatures of selection have pointed out that pleiotropy constrains the rate of adaptation, which clashes with theoretical and empirical expectations that immune systems need to adapt quickly to counter the rapid evolution of pathogens and parasites. As central questions in my lab evolve around the evolution of immune systems, we want to investigate why pleiotropy is so broadly maintained in immune systems if it could constrain adaptation. My lab will employ several complementary approaches that account for feedbacks across biological scales to study the impact of pleiotropy on host-parasite coevolution. In our first approach, we will integrate genomic data from insect and vertebrate model organisms to investigate selection pressures acting on pleiotropic genes, transcripts, and protein domains and define common features of pleiotropic signaling networks. At the same time, we will build computational models of signaling network evolution to test whether single-sided evolution or coevolution against parasites fundamentally alters the maintenance of pleiotropy in networks. Our third approach will employ experimental evolution to target the deployment of pleiotropic signaling networks during the insect (Tribolium castaneum) host response to manipulative parasites so that we can investigate whether pleiotropy alters coevolutionary outcomes at genotypic and phenotypic levels. The molecular evolution results from the first approach will help us build more realistic networks for the agent-based models, which will then provide salient hypotheses for the experimental evolution outcomes. In turn, the coevolution experiments will provide empirical insight into statistical signatures of selection and model predictions from the first two approaches. Together, these research avenues will provide insight into an array of fundamental questions about the extent of genetic pleiotropy among essential physiological processes, the influence of pleiotropy on coevolutionary dynamics, and the role of immune system architecture in host adaptation to parasite pressure. Gaining greater insight into the evolutionary forces that shape biological systems has important implications for predicting human pathogen evolution, understanding the origins of diseases like autoimmunity and sepsis, and designing therapeutic treatments that minimize side effects.