Duke University

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Signaling networks that control cellular behavior are highly dynamic and precisely coordinated in space and time. Rho family GTPases regulate diverse biological processes such as cell migration, proliferation and immune activation. The activity of these molecules is tightly controlled at the subcellular level and is observed with precise timing only in discrete regions of the cell. The Dbl family of guanine exchange factors (GEFs) are the main activators of RhoA GTPases. There are typically multiple GEFs present in a cell that can act on the same GTPase, and certain GEFs can interact with different GTPases. Recently, it has been shown that the activity of Dbl GEFs is also distributed at discrete regions in the cell and regulated with precise kinetics. Therefore, GEFs and GTPases form complex signaling networks that are tightly controlled in space and time. Traditional GEF studies typically rely on depletion, by knock down or knock out, or augmentation, by overexpression, of specific GEF activities. While informative, these approaches lack spatiotemporal resolution and could introduce biological artifacts due to possible compensatory effects in connected GEF/GTPase signaling networks. Therefore, to fully understand the biological roles of GEFs, new molecular tools are needed that allow the rapid and precise control of their activity in living cells. The goal of this proposal is to develop a high throughput platform that can be readily applied to engineer light-controlled inhibitors against the Dbl family of GEFs. These inhibitors will make possible the reversible inhibition of endogenous GEFs with second-level kinetics and at micron resolution in living cells. In Aim 1, three different approaches, that rely on computational modeling and high throughput library screening, will be tested to engineer molecules that bind with high affinity and specificity to Dbl GEFs and prevent their GTPase association. In Aim 2, engineered inhibitors will be fused to known optogenetic modules in order to allow the precise control of their activity by irradiation. In Aim 3, the optogenetic inhibitors will be studied by live cell microscopy to determine the experimental parameters that need to be fine-tuned in order to achieve efficient GEF inhibition in vivo. The utility of this platform will be demonstrated by engineering optogenetic inhibitors against three different Dbl GEFs that target the three major RhoA GTPases, Rac1, RhoA and Cdc42. The platform developed here is general and could be readily applied to develop molecular tools for the study of other Dbl GEFs. This proposal will thus facilitate the study of Dbl GEFs at unprecedent spatial and temporal resolution across diverse biological systems.

NIH Research Projects · FY 2025 · 2022-09

Epstein-Barr virus (EBV) was the first human tumor virus discovered over 50 years ago in the context of endemic African Burkitt lymphoma. However, we now know it is also a common herpesvirus that persists as a lifelong latent infection in virtually all adults worldwide. Early work in the field led to a model for EBV infection promoting B-cell lymphomas as evidenced by the growth transformation, or immortalization, of primary resting human B cells into lymphoblastoid cell lines (LCLs). In vivo, EBV latent infection is met with a robust cytotoxic T-cell response keeping most infected individuals protected from the oncogenic potential of the virus. As such, EBV-associated B-cell lymphomas occur at significantly higher rates in the setting of immune suppression. Studies of viral and cellular gene expression in EBV-infected cells in vitro and in vivo have led to a model of lymphomagenesis characterized by the full expression of EBV latency gene products. However, the phenotypes in bulk culture and tumor tissue lack the nuanced detail of cellular heterogeneity and the consequences of minor frequency phenotypes on cancer progression. Our recent single cell RNAseq experiments have characterized gene expression within individual EBV-infected B cells leading to an appreciation of cell fate trajectories and dynamic gene expression behavior of individual cells that we will integrate with human tumor analysis and mouse models of lymphomagenesis. It is our ultimate goal to define the importance of specific EBV-infected cell populations on the progression of B-cell non-Hodgkin lymphomas of the immune suppressed. In this proposal, we aim to define how EBV-infected cell heterogeneity, including innate antiviral restriction and plasmablast differentiation, impacts lymphomagenesis and can be exploited for therapy. Our central hypothesis is that EBV- infected B cells toggle between different states that can restrict or promote lymphomagenesis as well as render cells susceptible to virus-specific therapeutic intervention. We formulated our central hypothesis based on preliminary data including single-cell RNA sequencing of EBV-infected primary B cells early after infection and in LCLs as well as characterization of cell fate dynamics regulating plasmablastic differentiation and lytic reactivation. We also provide evidence supporting a recent clinical trial using the “kick and kill” strategy of promoting EBV lytic reactivation with histone deacetylase inhibition coupled with ganciclovir to kill lymphoma cells that activate viral kinases. Thus, the rationale for the proposed research is that understanding EBV regulation of infected B-cell fates will dissect mechanisms of pathogenesis and reveal new therapeutic avenues to target EBV-positive B-cell lymphomas. We plan to test our central hypothesis and complete the objectives in this proposal through the following three specific aims: i) to define the role of innate immune sensors and effectors in EBV-mediated immortalization and lymphomagenesis, ii) to determine the role of plasmablast differentiation in suppressing EBV-mediated lymphomagenesis, and iii) to define the mechanism by which HDAC inhibition promotes susceptibility of EBV+ DLBCL to killing by ganciclovir.

NIH Research Projects · FY 2026 · 2022-09

The U.S. is facing a biomedical workforce crisis, exacerbated by attrition starting from STEM majors in undergraduate training. The result is a lack of innovative solutions to advance health outcomes. Chronic kidney disease and end stage kidney disease represent a substantial medical and economic burden in the U.S., with annual management costs estimated at $120 billion. Duke University is uniquely positioned to address this critical problem by building a robust, and sustainable pipeline of future academic biomedical scientists. Our recently established Office of Physician Scientist Development (OPSD) offers a sustainable structure for mentorship, professional development, and research funding. By leveraging this institutional structure, we are well positioned to support the development, implementation, and evaluation of a program that links resources across the training spectrum to introduce undergraduate students to varied research career opportunities, while preparing senior trainees transitioning to faculty positions to become strong independent investigators skilled in mentoring. To address the biomedical workforce gap, we propose the Paired Undergraduate Mentoring Program (PUMP) in Uronephrology, which will recruit cohorts of undergraduate students and expose them to research skills-building and mentored research experiences in urology and nephrology. We will provide evidence-based professional development activities targeting both the student participants and the trainees who will serve as the day-to-day research project mentors. Duke PUMP will achieve our long-term goal, addressing barriers that limit the recruitment and retention of a uronephrology workforce, through intensive research experiences, structured triangular mentorship, and sustained virtual engagement. The program focuses on 1) implementing a mentored summer research enrichment program to develop interest in uronephrology research careers among a cohort of rising junior and senior undergraduates; 2) preparing next-generation uronephrology investigators to mentor by training graduate students and postdoctoral associates in mentorship and providing them critical experience leading student research projects; and 3) preparing a cohort of undergraduates for post-graduate training and entry into uronephrology research careers through sustained engagement that extends and enriches the summer research program experience. Successful implementation of the proposed program will lead to a scalable model for development of a robust pipeline for a biomedical research workforce across disciplines

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY / ABSTRACT The preclinical phase of late-onset Alzheimer’s disease (LoAD) is a crucial time period for diagnosis and intervention that is not well-modeled by the transgenic animal models commonly used in aging research. This lack of strong models exists despite the fact that LoAD accounts for well over 90% of Alzheimer’s disease (AD) cases and thus represents the bulk of the disease burden. At particularly high risk for LoAD are female carriers of the ε4 apolipoprotein allele, a risk that has been linked to the menopause transition (MT). Macaque monkeys well-model the preclinical phase of LoAD; this species recapitulates patterns of accumulation of amyloid and tau pathology seen with aging in humans, and shows accompanying memory impairment equivalent to mild cognitive impairment in humans. Furthermore, female macaques undergo the MT, and there is only one macaque apolipoprotein isoform, equivalent to the human ε4 allele; all female macaques are therefore at high risk for LoAD-like pathology. The macaque monkey thus offers a unique opportunity to study preclinical brain changes in aging and LoAD, in a species with a brain that is structurally and functionally very similar to that of humans. It has long been known that in humans during the preclinical phase of LoAD, amyloid and tau pathology show characteristic sites of origin and (particularly for tau) patterns of spread; the mechanisms behind these spatial features of the disease are not yet known. In order to understand the spatial neurochemistry of the aging primate brain, we will study peri- and post-MT female macaques, and age- matched male controls, delivering within-subject spatial atlases of metabolite levels (spatial metabolome), protein expression (spatial proteome), and extant pathology for the cortex and cerebellum. The metabolome and proteome will be obtained from the same samples, allowing trans-omic integration to understand spatial patterns of biochemical pathway activity across the brain. The histopathological data will come from the opposite hemisphere of the same individual. We will use spatial principal components analysis and a novel spatial adaptation of multiple correspondence analysis to identify individual biomarkers and combinations of biomarkers that account for spatial position in the cortex, and to identify biomarkers and biomarker combinations that are associated with observed (in an individual) and predicted (from the literature) spatial patterns of pathology. These crucial data will yield unprecedented insights into early aging and the critical prodromal phase of LoAD, offering possibility of identifying biochemical factors that confer risk or resilience upon local brain circuits. Factors promoting resilience are opportunities for intervention, while risk factors hold the possibility for early diagnosis and screening; both of these outcomes will be crucial if we are to understand and effectively address the most prevalent form of this devastating brain disease.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY The claustrum is a narrow, subcortical sheet of gray matter with interconnections across the cerebral cortex. Its irregular shape and deep location make it difficult to study with classical neurophysiological techniques. Recent genetic technologies such as optogenetics provide new hope for understanding the claustrum. They have advanced its study in rodents, implicating the claustrum in an array of sensorimotor and cognitive functions. Circuit-specific studies are needed to resolve how these functions map to the claustrum’s widespread connections. Such studies would be especially valuable in macaque monkeys, the animal model most homologous to humans. My overall goal is to translate viral technologies to monkeys and use them to study the role of a specific claustrum-prefrontal cortical circuit in visuo-saccadic behavior. During systematic testing of viral vectors in macaques, I identified a promising candidate for delivering opsin genes to the claustrum: the retrograde virus rAAV2-retro. When injected into the frontal eye field (FEF), a prefrontal cortical area involved in vision, movement, and cognition, rAAV2-retro constructs yielded strong labeling of FEF-projecting claustrum neurons. This finding provides a long-sought, unique opportunity to study claustrum neurons with circuit-level specificity in the primate brain. I propose to validate viral techniques in the claustro-FEF circuit and use them to test my overall hypothesis that the claustrum mediates competitive selection of the most behaviorally relevant stimulus via suppression of all other stimuli. To test my hypothesis, I have three integrated aims. In Aim 1, I will conduct anatomical tracing studies to elucidate the motif of connectivity between the claustrum and FEF. In Aim 2, I will use rAAV2-retro to express an inhibitory opsin in FEF-projecting claustrum neurons. Then I will use phototagging to identify those neurons and extracellularly record from them to characterize the signals sent from the claustrum to the FEF during a battery of saccade tasks. In Aim 3, I will use the same inhibitory opsins to selectively silence FEF-projecting claustrum neurons, allowing me to determine whether they are required for competitive selection of saccade targets. The claustrum is implicated in a wide range of neurological diseases and neuropsychiatric disorders. Thus, the results of the proposed work will help reveal how pathological changes to neural activity in the claustrum may contribute to brain disorders. In addition to its clinical relevance, the proposed work will significantly expand my doctoral training to include neural recording and optogenetic methods in non-human primates.

NIH Research Projects · FY 2025 · 2022-09

Abstract Peripheral tissue injury, which arises due to surgical trauma, can negatively impact brain function, particularly in older and/or frail patients that may already suffer from neurodegeneration and Alzheimer’s disease (AD). The blood-brain barrier (BBB) represents a key interface between the periphery and the brain with important roles in regulating neuroimmune interactions. Dysfunction of the BBB has been reported in many neurologic conditions, including AD and Alzheimer’s Disease Related Dementias (ADRD). BBB dysfunction during normal aging can also contribute to neuroinflammation and cognitive loss via altered transcellular permeability, trafficking of blood- derived factors in the brain parenchyma. BBB dysfunction and neuroinflammation are growing significant interest in postoperative complications such as delirium. Indeed, delirium is a form of ADRD that is associated with worse outcomes both in patients with and without neurodegeneration, and is especially common following major surgery such as orthopedic procedures. In this research, we will address why the aging brain is more susceptible to postoperative delirium and neuroinflammation by using a clinically relevant orthopedic fracture model in mice that displays various cardinal features of BBB impairment, glial dysfunction, and cellular senescence. Our central hypothesis is that senescent cell burden in aged individuals exacerbates systemic inflammation, hastens BBB breaching, and promotes neuroinflammation leading to delirium. We will test this hypothesis by combining a transgenic mouse model and pharmacological drug-mediated elimination of senescent cells. The latter will also lead to the validation of a clinically applicable therapeutic formulation/regimen to treat post-operative neuroinflammation and delirium with drugs (senolytics) currently tested in humans for other disorders, including AD. We have 3 objectives: 1) determine the role of senescent cells on systemic inflammation, BBB breaching, and neuroinflammation following surgical trauma, 2) examine the efficacy of senolytic drug, D/Q-mediated senescent cell clearance in preventing neuroinflammation, and 3) determine the reciprocal interactions between the blood-borne factors and the BBB using a microphysiological system. Together the studies will provide new insights into the effects of peripheral surgical trauma on the blood-brain interface, neuroinflammation, and cognitive impairments such as delirium. This is a highly significant step toward identifying pharmacologic targets to prevent delirium and improve outcomes after surgery in elderly patients.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Different magnetic resonance imaging (MRI) scanners and different acquisition parameters can produce very different images for the same patients. This is a significant issue when attempting to use MRIs in a quantitative manner. Multiple studies have shown promise of quantitative analysis of breast MRIs to diagnose breast tumors, predict patient outcomes, assess cancer risk, and even identify genomic signatures of cancers. However, the issue of inhomogeneity of images hampers the progress of the research and clinical implementation of these findings. In many cases one cannot utilize images from different sources to answer a research question. Furthermore, predictive models developed at one institution may not generalize to other institutions. While this is a well-recognized problem, there is currently no solution to it in breast MRI. Some valid efforts have been undertaken in order to address this issue for other organs, predominantly brain. However, the problem has not been solved for those organs neither and limited validation of the existing methods in practical contexts hampers the implementation. Breast is a non-rigid organ with highly variable composition making the harmonization of breast MRIs particularly challenging and making almost all prior harmonization methods developed for brain not applicable. Given the urgent need for harmonization in quantitative research, we propose three harmonization methods that allow for transforming an image acquired using one scanner setup to assume appearance of another scanner setup. We introduce important technical innovations to utilize cutting-edge convolutional neural networks for this task. Additionally, we propose a new approach to the question that has not yet attracted significant systematic consideration: what makes a harmonization algorithm successful or useful? We do not evaluate pixel-to-pixel match between the harmonized image and a reference image which is the typical approach. This approach is impractical in breast imaging since it requires ideally paired images, it does not deal well with expected image noise, and it does not inform about specific limitations of the evaluated harmonization method. We propose an evaluation framework that assesses harmonization algorithms in terms of different practical applications including radiomic analysis and deep learning. The study will be conducted in collaboration between a machine learning scientists (Duke and Yale), a breast MRI physicist (Cornell), a radiologist whose research focuses on MRI (Duke), and a biostatistician (Duke). The proposed harmonization and evaluation methods do not require fully paired data and do not make assumptions about tissue composition. Therefore, they will be applicable across other organs once implemented with appropriate data for the organ. All harmonization and evaluation algorithms along with the data will be made publicly available to spearhead further research on this crucial unsolved research topic.

NIH Research Projects · FY 2025 · 2022-09

Enter the text here that is the new abstract information for your application. Duke University School of Medicine has identified the lack of robust post-baccalaureate research training at our institution as a critical obstacle to the recruitment of candidates into graduate biomedical research programs. To address this barrier, we are developing a multi-track post-baccalaureate research scholars program: the Duke Preparing Research scholars In bioMEdical sciences-Post-baccalaureate Research Education Program (PRIME-PREP). Drawing on our nationwide network to identify and recruit candidates from, PRIME-PREP will seek to enhance students’ success in entering and completing research-focused doctoral degree programs. To ensure productive mentoring relationships, research preceptors have been identified based on their mentorship records and their established, funded independent research programs. Prospective mentors will complete mentor training before placement of scholars in their laboratories. This training not only serves the individual post-baccalaureate scholars, but also more broadly promotes a culture of effective and supportive mentoring necessary for the success of the next generation of biomedical scientists. PRIME-PREP Scholars will participate in a tailored professional development curriculum that prepares them for successful entry into PhD programs. In addition to peer networking within the PRIME-PREP cohort, scholars will have access to a community of peers, near-peers, and leaders participating in other tracks of the overarching Duke PRIME program, developed and funded independently of PRIME-PREP. The one-year PRIME-PREP will place students in laboratories, where mentored research will comprise 75% of their time. Professional development activities and workshops will comprise 25% of their time, with the 6-week summer bridge program at the beginning of the year constituting a large share of the total. By implementing and adapting evidence-based PREP frameworks, PRIME-PREP will place at least 75% of participants in PhD programs within two years of program completion.

NIH Research Projects · FY 2025 · 2022-09

Eating disorders (EDs) are serious psychiatric illnesses that disproportionately affect young women1, 2. Early intervention, when individuals show some signs and symptoms, but before behaviors become more automatic and entrenched can mitigate significant morbidity and mortality3-6. However, there are limited early ED interventions and ED treatment broadly suffers limited access and engagement7-10. Digital interventions have high potential to overcome access barriers and appeal to young people at greatest risk for ED onset2, 11, yet are undeveloped. Many digital interventions simply convert information or in-person activities to an online format or focus on screening and referral, and interventions are primarily based in traditional cognitive-behavioral therapy (CBT)10, 12. Few (if any) have taken a user-centered design approach to development or used gamification, which may increase engagement and learning through operant learning principles. In an initial proof of concept study, we piloted a novel digital, gamified intervention based on Acceptance and Commitment Therapy (ACT)13. Rather than the typical approach to the body weight concerns that underlie EDs, the intervention used gamification to experientially train body-image flexibility (BIF) (i.e., the ability to have distressing thoughts/feelings about the body, without unnecessary attempts to avoid or escape these experiences, and pursue other personally meaningful values or goals). This is a new direction with preliminary support, and would be a paradigm shift in early ED intervention, which has focused on changing or eliminating body-image distress. This Phased R61/R33 (NIH Stage 1A-B) takes a user- centered design and experimental therapeutics approach to further develop and optimize the intervention for young women in the US with ED symptoms. In the R61 Phase of the study, draft sessions are built with streamlined content, and enhanced graphics and interactive features, and we conduct iterative user testing to maximize acceptability and impact on BIF (Aim 1). We then use a multiple baseline experiment across participants to test the effect of the intervention (“FlexED”) on BIF, our targeted mechanism of change, and establish treatment dose (Aim 2). During the R33 Phase, we conduct a pilot RCT comparing FlexED to an online educational control. We test the effect of the intervention on BIF and the associated clinical benefit, and assess whether the intervention results in decoupling of body-image distress and behavior as an additional test of our mechanism of change (Aim 1). We also test a virtual body-Behavioral Approach task as an assessment of BIF in a personally meaningful or valued context that may be used in future investigations (Aim 2). Finally, we assess the acceptability of the final FlexED intervention, as indicated by retention (Aim 3). This proposal prepares for a larger trial with longer-term follow up, with the ultimate aim of establishing a cost-effective, widely available early intervention to decrease the societal impact of EDs1.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT Bone fracture is a very common injury, and there has been a dramatic increase in trauma-induced fractures with the increase in active lifestyle. Despite the regenerative ability of bone, bone injuries often suffer from delayed healing or non-unions. The prevalence of bone fracture and the cost of repair is on the rise primarily due to aging of the population. The profound negative impact on the patient’s quality of life and the economic burden of fracture treatment following trauma or age-related fragility fractures warrants the development of efficient and cost-effective fracture-healing adjuvants to accelerate the healing rate and improve the quality of healing. The proposed research focuses on biomaterial-assisted local delivery of adenosine to promote bone healing of aged bone tissues by rejuvenating the endogenous tissue environment and reparative cells. We hypothesize that local delivery of adenosine at the injury site will induce a pro-regenerative immune environment and promote regeneration of aging bone tissues through enhanced osteoblastogenesis and angiogenesis. These hypotheses will be tested through the following aims. Aim 1 will determine the effect of local delivery of adenosine to promote bone regeneration in aged mice by using two injury models: transverse tibial fracture and critical-sized segmental femoral defect. Aim 2 will determine the role of adenosine delivery on immunomodulation leading to enhanced bone regeneration. Successful completion of the proposed studies will have a significant impact in public health by establishing a new therapeutic intervention for promoting bone regeneration.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Systemic inflammation triggered by surgical trauma can negatively impact brain function, particularly in older adults and/or frail patients with pre-existing dementia. One frequent neurologic complication for these patients is delirium, an acutely debilitating change in brain function that precedes an adverse prognosis. Indeed, patients that develop delirium superimposed on dementia (DSD) have significiantly worse outcomes with mortality rates as high as 92% two years after surgery, compared to a 7% post-surgical mortality in patients without dementia or delirium. Damage to the blood-brain barrier (BBB), a key interface that regulates neuroimmune interactions between the periphery and the brain, may play a role in DSD. Using a clinically-relevant mouse model of delirium- like behavior as a result of orthopedic surgery, we have identified a prominent role for the innate immune response in promoting BBB dysfunction, and neuroinflammation. Notably, BBB breakdown has been reported in many neurodegenerative conditions, including Alzhemeir’s disease (AD) as well as during aging via altered transcellular permeability and trafficking of blood-derived factors into the brain parenchyma. Yet, the mechanisms by which surgery impacts the BBB and the specific role(s) of vascular dysfunction in DSD remain unknown. Our overall objective is to identify postoperative mechanisms for BBB dysfunction in DSD. Our central hypothesis is that systemic factors impair structure and function of discrete brain vasculature and BBB leading to neuroinflammation, neurodegeneration, and delirium-like behavior in AD mice after surgery. We propose two Specific Aims: 1) to characterize how the postoperative systemic milieu impacts the BBB using organ-on-chip technology comprised of μSiM (microphysiologic system with nanoporous silicon membranes) populated with human iPSCs; and 2) to define cellular and molecular mechanisms mediating DSD-induced vascular dysfunction after surgery. Feasibility for these models and techniques has been established in the applicants’ hands. In this innovative approach, organ-on-chip technology will complement unbiased spatial profiling of vascular changes in the brain of dementia-prone mice with delirium-like behavior. The rationale for the proposed research is that successful completion will expand our understanding of how surgery affects the blood-brain interface, and will provide new molecular mechanisms of relevance to delirium, and neurodegeneration. Such knowledge is highly significant because it will implement new technologies to investigate immune-vascular interactions and inform the advancement of safe therapies to limit postoperative neurocognitive complications in vulnerable patients.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT. Breast cancer screening programs suffer from false positive mammograms, unnecessary biopsies, overdiagnosis, and overtreatment. A major contributor to the poor performance of screening mammography is the diagnostic and prognostic uncertainty of mammographically detected calcifications. Breast calcifications represent a biological continuum from benign disease to ductal carcinoma in situ (DCIS) to aggressive cancer. Radiologists struggle to correlate their imaging appearance with the underlying pathology and roughly two-thirds of biopsied calcifications return with a benign pathology. Although calcifications evolve dynamically over time, the current management strategy relies heavily on the static appearance of calcifications from the most recent mammogram. Most women in screening programs have multiple mammograms, yet this temporal information is consistently underutilized in clinical decision making. There is thus an urgent need to quantify the dynamics of calcifications from serial mammograms, and to characterize the relationship between calcification trajectories and disease biology. In the absence of such innovation, increasingly sensitive screening modalities are expected to further increase the burden of unnecessary diagnostic work-up and breast cancer overdiagnosis. The central hypothesis of this proposal is that dynamic imageable and tissue biomarkers contain actionable diagnostic and prognostic information about mammographic calcifications. The use of established diagnostic imaging (mammography) in conjunction with investigational imageable biomarkers will enable testing of this hypothesis. Key to this proposal will be the creation of a large database of retrospectively and prospectively collected cohorts of patients with serial mammograms, tissue samples and clinical outcomes. This proposal will consist of three specific aims: (1) Develop a static model of breast calcifications to improve the clinical performance of mammography screening; 2) Develop a dynamic model of breast calcifications to predict histopathology and DCIS prognosis; and 3) Combine the dynamic calcification model with tissue-based biomarkers of the underlying evolutionary dynamics to delineate DCIS prognosis. The proposed research is highly innovative because it adds the temporal dimension to computer-assisted classification of mammographic calcifications, yields a joint characterization of calcification growth trajectories and lesion biology, and develops dynamic risk models to predict invasive progression in women undergoing active monitoring for DCIS. This proposal will be co-led by Dr. Grimm (breast radiologist) and Dr. Ryser (mathematical modeler) supported by a highly collaborative multidisciplinary team with expertise in cancer biology, computer vision, and surgical oncology. The overall objective of this proposal is to develop a dynamic imageable biomarker that delineates lethal cancer from non- lethal disease by leveraging the temporal dimension of serial mammograms. Ultimately, the long-term goal of our work is to better identify which calcifications to biopsy (reduce unnecessary biopsies), and if pre-invasive DCIS is found, to predict whether it will remain indolent or progress to lethal cancer (reduce overtreatment).

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT This application for the Paul B. Beeson Emerging Leaders Career Development Award in Aging (K76) is for Dr. Corey Simon, a physical therapist and geriatric pain researcher specializing in low back pain. Low back pain is one of the world’s most disabling conditions for older adults, with more than 75% reporting persistent disability 1-2 years after onset. A novel disability mechanism among older adults with low back pain is high stress reactivity, which is an acute physiologic response characterized by abnormal changes in blood biomarkers after stressful encounters. High stress reactivity is linked to poorer health outcomes including disability in other chronic conditions; and accumulating research suggests high stress reactivity is mediated by abnormal thoughts and feelings and is modifiable through biobehavioral interventions. However, stress reactivity research in older adults with low back pain is lacking. This proposal in an innovative 5-year research program that builds upon Dr. Simon’s pilot study demonstrating exciting preliminary associations between stress reactivity, physical function, and psychological distress. This proposal will utilize novel laboratory stress reactivity tests, patient-reported outcomes, and qualitative interviews to: 1) Identify and quantify stress reactivity in older adults with low back pain; and 2) For the purpose of reducing disability risks, develop a biobehavioral intervention that targets high stress reactivity in older adults with low back pain. This program will test his central hypothesis that older adults with low back pain and high stress reactivity are at greater risk for disability due to negative thoughts and feelings and poor coping strategies. In addition, this program will provide Dr. Simon with advanced career development in stress reactivity science, behavioral intervention development, and research leadership. Upon completion of this program, Dr. Simon will be poised to facilitate the development of an NIA-funded clinical trial (R34/R01) to test the efficacy of his innovative biobehavioral rehabilitation intervention for older adults with low back pain that targets high stress reactivity. Collectively, this program is the next step in Dr. Simon’s long-term goal to become an international leader in the development of targeted interventions to eradicate disability in older adults with low back pain.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Although many people are familiar with the staggering statistic that up to 30% of Americans suffer from chronic pain, a less-known fact is that most individuals with chronic pain suffer from more than one comorbid pain condition. Pain conditions which frequently co-aggregate have come to be known as chronic overlapping pain conditions (COPCs). Co-aggregation of multiple chronic pain conditions frequently results in negative side effects in addition to pain, including fatigue, sleep deficits, cognitive impairment, functional impairment, and mental health conditions such as depression, anxiety, and even suicidal ideation. Given the complexity of these side effects and their impact on patient well-being, COPCs have worse health outcomes compared to patients with a single chronic pain condition. In addition, the majority of individual COPCs such as temporomandibular joint disorders (TMD), fibromyalgia (FM), and migraines have a higher prevalence in biological females than in biological males, and their coaggregation is likewise higher in females. Thus, it is imperative that additional research investigations be undertaken which explores the mechanistic determinants of COPCs in patients with multiple comorbid conditions, and which critically evaluates the underlying mechanisms in biological males and females. We have developed methods that use clinical, biological, and psychological patient variables to group patients with COPCs into distinct patient clusters: (1) an "adaptive" cluster, which is free of hypersensitivity and psychological distress; (2) a "pain-sensitive" cluster, which has increased pain sensitivity to pain stimuli, but lacks pain-related comorbidities; and (3) a "global symptoms" cluster of patients which has increased sensitivity to pain, along with multiple symptoms of depression and anxiety, and other widespread symptoms. By separating phenotypically distinct COPC patients and analyzing these cohorts as separate entities individually in males and females, we believe we can better understand these conditions and create a more personalized approach to patient care, which will ultimately improve our ability to treat patients with multiple chronic pain conditions. Our overall hypothesis is that patients within each cluster will exhibit a greater degree of similarity in their cellular and molecular makeup compared to patients in other clusters, and we can exploit these cluster-related differences to identify biological markers and treatment approaches that are personalized for each patient group. To test this hypothesis, we will employ proteomic, transcriptomic, and preclinical screening approaches to ascertain molecular and cellular mediators of pain in individual patient clusters and in each sex. In doing so, this project will yield new mechanistic insights into the divergent pathophysiological mechanisms that give rise to pain and its associated comorbidities in males and females.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Locally advanced rectal cancer, defined by spread to lymph nodes or extension through the full rectal wall thickness, is diagnosed in over 15,000 US patients yearly. Standard treatment for locally advanced rectal cancer in the US involves chemotherapy and radiation therapy (chemoradiation, CRT) prior to surgical removal of the entire rectum. Patients may experience undesirable post-surgical consequences such as permanent colostomy or altered anorectal function. In light of this, much ongoing research focuses on strategies to avoid surgical intervention in the subset of patients who might be cured with CRT alone. At the time of surgery, 10- 30% of tumors are eradicated by CRT (i.e., they demonstrate a pathologic complete response) while other tumors show little to no response. This variability in response is incompletely understood, but likely relates to the fact that one tumor may contain numerous different tumor cell subpopulations, all with different genotypes and epigenetic (or non-genetic) features. These diverse subpopulations and variable genetic/epigenetic features can interact, resulting in a dynamic and evolving tumor cell network that is poorly understood. In an increasing number of cancers, there is evidence supporting the emergence of treatment-induced evolutionary “traps,” or “collateral sensitivities”, where resistance to one therapy results in sensitivity to another therapy. Whether this occurs in rectal cancer, and how to leverage it into treatment strategies, is unclear. To address these questions, it is crucial to select a representative experimental model. Thus, the current proposal leverages an already-established co-clinical pipeline from which patient derived tumor samples are used to generate paired, patient-specific organoid and xenograft models. Two broad analytic approaches will be undertaken to identify trends in CRT-induced tumor evolution and elucidate potential synergistic approaches to improve CRT response. Specific Aim 1 will dissect CRT-induced changes using genomic, epigenomic, and single cell profiling techniques, including an advanced method combining single cell RNA-seq with lineage tracing. Specific Aim 2 employs an innovative iterative paired CRISPR/high throughput drug screen approach to identify evolving collateral sensitivities in CRT-treated rectal cancer. To validate the findings in both Aims, advanced patient-derived rectal cancer organoid and xenograft models will be utilized. Research, scientific instruction, and career development will be supported by an expert panel of mentors and collaborators and a structured training plan. Successful completion of both Aims will be promoted by the expertise and existing screening and genomic infrastructure in the co-mentors’ laboratories and leverages a unique co-clinical platform already established by close institutional collaborators. This research will establish a patient-derived platform for interrogating CRT-induced tumor evolution, establishing a scientific niche to facilitate success during the transition to scientific independence.

NIH Research Projects · FY 2025 · 2022-09

Project Abstract for NIH R35 (MIRA): The discovery and development of new methods for the efficient synthesis of N-containing and F-containing chemical building blocks is an important goal in organic synthesis as a large number of pharmaceuticals and other bioactive molecules contain these atoms within a diverse set of chemical functional groups. More rapid and/or selective assembly of known motifs, and moreover the preparation of new chemical landscapes, requires innovative approaches to drug-like scaffolds, including the discovery of new reagents and new catalysts/catalytic strategies. The current goals of this project fall under three main focus areas. 1) We will develop 2-azatrienes, a novel class of enamine umpolung reagents, for myriad catalytic enantioselective approaches towards chiral amines. Representative reactions that will be developed include 6,3-, 6,5- and 5,6-hydrofunctionalizations including reductive couplings with carbonyls and imines, hydroalkynylations, and hydroarylations. Azadienes generated from 6,5- and 5,6-hydrofunctionalizations of the azatriene reagents may be utilized in myriad downstream reactions, including other catalytic processes, thereby providing a diastereodivergent avenue towards highly complex chiral amines through sequential catalysis. 2) We will expand upon our prior work in enantioselective transformations of 2-azadienes, the first class of enamine umpolung reagents developed in our laboratory. Examples include reductive couplings with aromatic heterocycles, such as quinoline N-oxides, catalytic enantioselective fluorofunctionalizations with 4,4-difluoro-2-azadienes, cascade desymmetrization reactions, and reductive [3+2]-cycloadditions. 3) We will develop catalytic remote C–C and C–B coupling reactions that result in the loss of a halide from a trifluoromethyl group or H-atom abstraction from a difluoromethyl group to deliver difluorocarbons in a number of settings. In one case, we will carry out borylation or enantioselective alkylation of a 3-trifluoromethylpyridine scaffold to yield medicinally important and highly functionalized 3- (difluoromethyl)pyridines. In another area, we will execute a radical hydrogen atom abstraction of 4- difluoromethyl-2-azadienes to furnish a difluoro-2-azapentadienyl radical, which then may be engaged in catalytic cross-couplings to furnish chiral allylic amines bearing a difluoroalkene unit. Together, these undertakings will enable new chemical space for drug discovery to be obtained readily and with great diversity from simple reagents. We will access more established N-containing motifs more quickly and with greater levels of regio/stereocontrol compared to known approaches because of the invention of new reagents and the novel reactivity that they embody.

NIH Research Projects · FY 2025 · 2022-09

Title: Precision editing of neural circuits using engineered electrical synapses Pioneering approaches including optogenetics and designer receptors exclusively activated by designer drugs (DREADDs) enable the direct modulation of the activity of individual genetically defined cell types. Nevertheless, it remains a fundamental challenge to selectively regulate the hallmark feature of neural circuits: the interface between two specific brain cells. To address this challenge, we have created a new approach, Long-term integration of circuits using Connexins (LinCx), that employs a novel pair of engineered connexin hemichannels to directly modulate genetically defined neural circuits. When each member of the hemichannel pair is expressed in two different cell(s)/cell-types that compose a circuit, they engage in heterotypic docking (docking with each other) and an electrical synapse is constituted between the two cells. These pair of hemichannels is engineered 1) to prevent them from engaging in homotypic docking (forming electrical synapses with themselves), and 2) to disrupt them from docking with other connexin hemichannels endogenously expressed in the mammalian brain. Finally, 3) the hemichannel pair exhibits rectification. Together, these three properties confer LinCx with unprecedented spatial-, temporal-, and context precision, enabling the precise editing of neural circuits. We propose to deploy LinCx across model organisms. We will determine the impact of LinCx neuromodulation on neural circuit physiology and emotional behavior. We will also test whether LinCx modulation is sufficient to restore normal behavior in animal models of psychiatric disorders. Successful completion of these high-risk experiments will yield a new method for long-term circuit editing to regulate emotional states in preclinical models. In the future, LinCx can be integrated with emerging viral tools that enable systemic delivery of genetically encoded proteins to specific brain cell-types. Thus, LinCx also has an attainable path to human translation for ameliorating devastating psychiatric disorders.

NIH Research Projects · FY 2024 · 2022-09

Reactive chemicals such as chlorine and phosgene pose a grave threat to respiratory health. These agents were used in warfare, with a significant risk of diversion for terrorist attacks, and frequently cause injuries due to accidental release. Little progress has been made in developing countermeasures, with supportive treatment remaining standard of care. In this application, we hypothesize that inhalation of chlorine and phosgene damage different subsets of pulmonary cells critical for maintenance of epithelial and endothelial barriers, gas exchange, and tissue recovery. Our hypothesis is based on preliminary studies in rodent and porcine chlorine exposure models in which we discovered a novel lung repair mechanism relying on submucosal lung cells that repopulate and differentiate into new epithelia. In contrast to chlorine, phosgene initially spares upper airway cells and primarily damages pulmonary endothelial cells, including newly discovered alveolar endothelial cell subtypes, specialized aerocytes (“aCap”) and general capillary (“gCap”). Comparing mice and pigs, we noted that the identity and location of cell populations in the pig lung more closely resemble the human anatomy. The following specific aims are designed to comprehensively analyze the short- and long-term effects of chlorine and phosgene on lung cell populations in rodents and pigs: Aim 1. Monitor molecular identities and temporal dynamics of pulmonary cell populations after exposures to chlorine or phosgene and during recovery; Aim 2. Visualize pulmonary epithelial and alveolar cells and structures after chlorine or phosgene injury using thick slice microscopy; Aim 3. Compare effects of ventilator and oxygenation support and positional maneuvers on pulmonary cell survival after phosgene or chlorine exposure

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT The ultimate goal of the proposed studies is to contribute to ending the HIV/AIDS epidemic. It has been four decades since the start of the HIV/AIDS epidemic and a protective vaccine or functional cure has been elusive. In 2020, there was an estimated 37.6 million people living with HIV. Despite highly active anti-retroviral therapies, hundreds of thousands of people still die from AIDS-related diseases and millions of new infections continue to emerge. Thus, finding a way to end this pandemic remains a global priority. The overall goal of the Consortium for Innovative HIV/AIDS Vaccine and Cure Research (CIAVCR) is to develop an effective combined immunotherapeutic regimen for HIV-1 prevention and cure using the non- human primate (NHP) model that has a direct path to the clinic for use in humans. There are two FOCI proposed in the CIAVCR: In FOCUS 1, our overall goal is to demonstrate the correlates and mechanisms of protection for a protective vaccine, and the role of vaccine-induced immune responses in selecting and limiting the latent reservoir. The benefits of using novel mRNA constructs to deliver immunogens that can elicit both humoral and cellular responses will be evaluated. In FOCUS 2, our overall goal is to determine the role of vaccine-induced immune responses to 1) control HIV-1 infection by reducing the size or eliminating HIV-1 reservoirs, and/or 2) delay plasma virus load rebound. The protective vaccines studied in FOCUS 1 will be tested to define the mechanisms of vaccine-induced B and T cell responses in clearing HIV-1 reservoirs. Additionally, novel therapies will be combined with the vaccines to augment clearance of HIV-1 reservoirs. In both FOCUS 1 and 2, analysis of the breakthrough and latent reservoir Env sequences will inform the design of new vaccine boosts for an improved protective vaccine regimen that can also limit rebound viruses. The NHP-SHIV Centralized Research Resource (CRR) will support the NHP studies in FOCUS 1 and 2 to investigate the effectiveness of vaccine-induced polyfunctional responses and novel immunotherapies in clearing HIV-1 reservoirs. These studies will use innovative barcoded-SHIVs to determine the effect of vaccine-induced responses on eliminating the viral reservoirs, and to evaluate the quantity and quality of viruses that are reactivated by latency reversing agents (LRAs) following treatment interruption. The Management and Operations Support Unit (MOS) will coordinate the scientific and administrative activities of this CIAVCR Program to ensure that the FOCI and NHP-SHIV CRR function cohesively. By the end of this grant, we expect to have designed a combined preventive and therapeutic approach to effectively protect from infection and eliminate viral reservoirs—a strategy for effectively impacting the HIV/AIDS pandemic.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Autism Spectrum Disorder (ASD) is a neurodevelopmental disease affecting almost 2% of children in the US alone. Despite its genetic and clinical heterogeneity, recent systems biology and genomics studies demonstrated that ASD converges on a specific set of cellular pathways. Epigenetic regulation and synaptic signaling emerged as the two most prominent pathways in ASD, with many high-confidence genetic risk factors and dysregulated genes involved in these processes. This observation prompted a hypothesis that epigenetic dysregulation leads to improper neuronal circuit development and function, which has been demonstrated in mouse models of epigenetic regulators recurrently mutated in ASD, such as CHD8 (Chromodomain Helicase DNA Binding Protein 8). However, the exact epigenetic changes, cell types they affect and the neuronal circuitry changes resulting from epigenetic dysregulation in ASD are unknown. Recently, single-cell genomics approaches, including single- cell RNA sequencing and single-cell ATAC sequinning, offered unprecedented new level of detail of cellular and molecular composition of the brain, as well as processes underlying its development. In my postdoc, I applied single-nucleus RNA sequencing to human post-mortem cortical tissue from ASD patients to gain insight into the molecular changes associated with ASD in specific neuronal and glial subtypes. One of the most important insights from this work is the implication of upper-layer cortical neurons as the cell type most affected by ASD- associated transcriptional changes. This observation raises questions about the origin and functional effects of such changes on specific neuronal circuits. As part of the Aim 1 of my K99 proposal, I will test the hypothesis that gene expression changes in ASD are driven by changes in epigenetic states of specific cell types. To that end, I will perform a joint RNA-seq and ATAC-seq profiling of neocortical tissue of ASD patients and controls to identify cell type-specific epigenetic changes. Then, I will develop and test a high-throughput synaptic tracing technique by combining barcoded rabies virus library with single-nucleus RNA sequencing (Aim 2 of K99 phase). Finally, using the training, tools and preliminary data from the K99 phase of my proposal, I will launch an independent research project that focuses on investigating cell-type specific epigenetic and neuronal circuitry changes in the Chd8+/ mouse model during development (R00 phase). I will first apply the joint RNA-seq/ATAC- seq protocol to study epigenetic changes in specific cell types during development caused by the loss of one of Chd8 alleles. By crossing the Chd8+/ mouse with reporter lines expressing Cre recombinase in specific neuronal subtypes, such as upper-layer cortical neurons (Cux2-Cre), I will be able to use the barcoded rabies virus library and single-nucleus RNA-seq to identify changes in specific components of cortical circuitry as the result of Chd8 haploinsufficiency. I believe that the K99-R00 award will allow me to form a unique research direction and establish myself as a successful independent investigator in the area of autism and single-cell genomics.

NIH Research Projects · FY 2025 · 2022-09

Early treatment of autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD) can improve long-term outcomes, but only when at-risk children are identified promptly and accurately. Early risk factors common to both conditions (e.g. premature birth, perinatal complications) are collected during routine care and are accessible in the electronic health record (EHR), but have not been used at scale to stratify risk or improve early screening. The central premise of the proposed research is that the EHR contains information that can help identify children at risk for ASD and ADHD early in development, but translating this information into clinically actionable measures requires new predictive modeling methods. Our recent NIMH-supported research provides initial support for this premise. We have discovered that children later diagnosed with ASD and/or ADHD have distinctive patterns of early health system utilization, and that EHR data acquired by age 1 already contains information predictive of later ASD risk. Building on this work, the proposed research will develop and deploy ASD and ADHD risk prediction models that can directly inform clinical decision-making. First, we propose to develop methods and prediction models that will optimize prediction performance, ensure predictions are not affected by disparities in utilization, and surveil model-predicted risk over time to identify children as early as possible without loss of accuracy. Second, we propose to take critical steps toward clinical use by prospectively evaluating these models, deploying them in the Duke University Health System (DUHS) EHR, and working with providers to develop and evaluate a prototype EHR dashboard that presents ASD and ADHD risk and projected follow-up length to inform clinical decision-making. Career development goals will allow the candidate to develop expertise needed to establish an independent, transdisciplinary research program in machine learning for pediatric mental health. Career training will be tightly integrated with the proposed research and emphasize deployment, evaluation, and implementation of risk prediction models for ASD and ADHD. In the proposed study, DUHS EHR data will be accessed through a high-quality, empirically validated data pipeline for all children born after 10/1/2006. Data elements will include diagnosis and procedure codes, lab measures and vitals, clinical notes, and encounter details. Diagnoses will be identified using ICD-10-based criteria previously validated at DUHS. The study will begin with creation of a secure, regularly updated diagnosis risk prediction database to facilitate a smooth transition to EHR deployment. Activities will focus on methods and model development in years 1-2 followed by development, deployment, and evaluation of the risk prediction dashboard beginning in year 3. This research will dovetail with concurrent and subsequent work exploring novel ASD screening methods and model-guided interventions at DUHS, including patient- and provider-centered early intervention strategies. The proposed career development will support the PI’s transition to independence and health data science initiatives at Duke.

NIH Research Projects · FY 2025 · 2022-09

An unanswered fundamental question in andrology is how cells recognize and respond in a different manner to different levels of physiological and synthetic androgens. It is assumed that since androgens differ primarily in their relative binding affinity for the androgen receptor (AR) that they are distinguished by how well they enable the formation of similar receptor-coregulator complexes and that this manifests as a quantitative continuum of the same responses. However, leveraging compelling new data we propose the alternate, albeit not mutually exclusive, possibility that androgen dose regulates the relative abundance of AR monomers and dimers in cells and that these forms of the receptor have different coregulator binding preferences resulting in different biological outputs. Indeed, using in vitro systems that model exposure from castrate (low dose; LD) to eugonadal levels and above (high dose; HD), we have determined that the global changes in chromatin architecture, transcription factor cistrome, and gene expression in cells are substantially different, with the changes induced by LD androgens (monomeric AR) being associated with cell proliferation and HD androgens (dimeric AR) inducing a program associated with a differentiated phenotype. A similar distinction in response to androgen levels was observed in vivo. Further, we made the surprising observation that both LD and HD androgens facilitate an AR dependent, non-genomic activation of mTOR but that the resulting translational outputs are different, such that LD but not HD androgens facilitate increased translation of mRNAs encoding key cell cycle proteins (i.e. E2F1, FOXM1). Importantly, we have identified high affinity AR ligands that do not allow receptor dimerization and have shown that their actions phenocopy those of LD androgens. Thus, by using receptor oligomerization state as a biosensor cells are able to respond differently to different levels of androgens; a process that can be exploited in the development of new AR modulators for the treatment of cancer and other androgenopathies. Hypothesis: Androgen receptor expressing cells possess biochemical mechanisms that enable them to manifest qualitatively distinct biological responses to different exposure levels of androgens enabling the same hormone to exhibit different activities in the same cell. Aims: (1) Define the mechanism(s) that enable cells to sense and respond to different levels of androgens, (2) Elucidate the mechanisms by which androgens activate mTOR and regulate mRNA translational specificity, and (3) Use small molecule-based approaches to explore the physiological and pathological importance of pathways and processes that enable cells to respond differently to different levels of androgens. Impact: In addition to probing the pharmacology of AR this study will formally test the “coregulator hypothesis” that differential engagement of functionally distinct coregulators allows the same ligand to exhibit different activities in and between cells. This will also enable the establishment of a conceptual framework that will inform the mechanisms that determine the molecular pharmacology of other ligand-regulated nuclear receptors.

NIH Research Projects · FY 2024 · 2022-09

Impaired fertility is a highly distressing, under-addressed, late effect of cancer and cancer treatment among female adult survivors of childhood cancer. Many female survivors are uncertain about their fertility status and report unmet fertility information needs and fertility-related distress. For those who desire biological children, overestimation of risk for impaired fertility may cause unnecessary emotional and relational distress. Underestimation may lead to a missed opportunity to pursue having biological children due to factors such as premature ovarian insufficiency. Yet, to date, fertility-related information needs and distress remain largely unaddressed in this population. Following the ORBIT Model for behavioral intervention development, the proposed study seeks to design and refine a behavioral intervention to address unmet fertility-related information needs and fertility-related distress among female adult survivors of childhood cancer (aged 18-44). Phase I of the study will involve conducting individual interviews with female adult survivors of childhood cancer (N=20) and medical providers who care for this population (N=10) to inform the development of a preliminary, manualized, intervention. Based on existing literature and feedback from experts in behavioral interventions for cancer survivors, adult survivors of childhood cancer, and infertility, it is anticipated that an intervention combining strategies from Patient Activation Theory and Acceptance and Commitment Therapy will be developed. Intervention development is flexible and will be tailored based on feedback received from stakeholders. In Phase 2, preliminary intervention content will be delivered to a small sample of the target population (N=30). Feasibility and acceptability, as well as examination of pre- to post-intervention patterns of change in intervention targets (primary: fertility health knowledge, fertility-related distress; secondary: psychological flexibility, patient activation, and self-efficacy), will be assessed and utilized to further refine the intervention (e.g., intervention strategies, intervention length, and delivery modality). In line with the National Cancer Institute's priority research area focused on cancer survivorship, the overall goal of the fellowship is to support Dr. Stalls to develop expertise that will enable her to become a leading clinical researcher in the development and evaluation of behavioral interventions to address reproductive and sexual health late effects among cancer survivors. To do so requires a unique combination of knowledge and skills in the areas of, 1) Reproductive and Sexual Health, 2) Behavioral Intervention Development and Evaluation, and 3) Professional Development. Training in these areas will be accomplished through guidance from expert sponsors and collaborators as well as the many resources and training opportunities offered at Duke University. Completion of this fellowship would not only advance the survivorship literature in this area and propel Dr. Stalls toward her career goals, but help address the significant gap in well-trained clinical researchers who are equipped to address these highly personal and time-sensitive reproductive and sexual health late effects of cancer and cancer treatment.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT: Ultra-processed foods (UPFs) account for over half of total caloric intake in high-income countries and are strongly linked to obesity, diabetes, cardiovascular disease, and premature death. However, current methods for assessing processed food intake rely on subjective recall tools that are prone to misreporting and require time-intensive manual classification of thousands of foods into broad processing categories. These limitations make cross-study comparisons difficult and impede robust links between UPF consumption and disease risk. The absence of objective biomarkers for UPF consumption, therefore, continues to limit our ability to establish definitive associations between diet and health outcomes. To address this challenge, we will extend the capabilities of FoodSeq as a novel, cost-effective genomic biomarker of UPF consumption. Our prior and ongoing work has demonstrated that targeted DNA sequencing of stool can reliably track a broad range of plant species intake, illuminate dietary compliance in controlled feeding trials, and reveal new hypotheses regarding industrial ingredient usage in the globalized food supply. By analyzing the plant DNA in stool, FoodSeq provides an objective measure of dietary exposure that circumvents many of the limitations of standard dietary assessment instruments. Building on this foundation, we will here assess whether the co-occurrence of DNA from three sentinel crops (corn, soy, and wheat) serves as an objective, stool-based biomarker of ultra- processed food intake in two Specific Aims: (Aim 1) We will use FoodSeq to systematically survey a representative array of UPFs, including cereals, snacks, frozen meals, and common industrial ingredients (e.g., corn starch, soy protein isolate, wheat gluten, soy lecithin), to confirm that plant DNA remains detectable despite heavy processing. We will then test the sensitivity, specificity, dose-response, and reproducibility of this co- occurrence signature in two controlled feeding trials: one contrasting a high-UPF diet with a minimally processed diet over multiple weeks, and another involving an intervention arm supplying 81% of daily energy from UPFs. (Aim 2) We will evaluate the broader predictive utility of our FoodSeq-based UPF biomarker in two large, well- characterized longitudinal cohorts: the Modeling the Epidemiologic Transition Study (METS, n=2,500) following adults in five countries at different stages of economic development, and the Nurses’ Health Study Microbiome Project (NHS Micro-N, n=5,000). We will compare the performance of our genomic biomarker with existing UPF indices (e.g., PEI-UPF, NOVA, Healthy Eating Index) and test for associations with cardiometabolic risk factors (BMI, blood glucose, type 2 diabetes, hypertension). We will also examine whether combining FoodSeq with survey-based approaches enhances risk prediction in these populations. Ultimately, by enabling objective, large-scale assessment of UPF exposure, this research has the potential to reshape nutritional epidemiology and support the development of more effective policies to reduce the global burden of diet-related disease.

NIH Research Projects · FY 2024 · 2022-09

PROJECT ABSTRACT Small cell lung cancer (SCLC) is a fatal neuroendocrine lung tumor that is challenging to treat due to early metastasis, rapid growth, and a lack of easily targetable driver alterations. For the last ~40 years, SCLC has been treated primarily as a single disease in the clinic with combination, platinum-based chemotherapy that offers a median survival of only ~10-12 months. It is imperative to better understand SCLC biology to enable development of novel treatment strategies that effectively prolong patient survival. SCLC tumors amplify or overexpress one oncogenic MYC family member: MYC, MYCL, or MYCN. MYC-high SCLCs are metabolically distinct from MYC-low, and have specific and targetable metabolic vulnerabilities. The most effective therapeutic strategy for treatment of MYC-high SCLCs in preclinical trials is deprivation of circulating arginine by pegylated arginine deiminase (ADI-PEG20). MYC-high SCLCs are particularly sensitive to ADI-PEG20, because they lack the enzyme argininosuccinate synthetase 1 (ASS1) that catalyzes de novo synthesis of arginine by the urea cycle. Still, SCLC tumors eventually develop resistance to ADI-PEG20 (ADIR) that corresponds with re- expression of ASS1. Upon ADIR, tumors acquire secondary metabolic dependencies that may be targeted to prolong ADI-PEG20 response and patient survival. Preliminary data show that ADIR SCLC depends on serine and one-carbon (1C) metabolism, which can be targeted with anti-folates. Preliminary data also delineate candidate transcriptional regulators that may govern ADIR in SCLC. Activating transcription factor 4 (ATF4), a stress-responsive transcription factor, is one predicted upstream regulator of gene programs enriched in ADIR vs naïve SCLCs—determined by bulk and single-cell RNA sequencing. ATF4 is induced upon acute arginine deprivation in SCLC and continues to be expressed with its target genes during ADIR. Here, the applicant will employ a single-cell RNA-seq-derived model of SCLC response to ADI-PEG20, metabolite profiling, in vivo isotope tracing, and CRISPR-based gene editing to interrogate whether ATF4 governs ADIR. The hypothesis for this research is that ATF4 drives ADIR by enhancing serine and 1C metabolism in an ASS1-dependent manner. Experiments will be performed in two specific aims to test whether ATF4 governs: 1) the sensitivity of MYC-high SCLCs to ADI-PEG20, and/or 2) the sensitivity of ADIR SCLCs to 1C metabolism inhibitors. Knowledge gleaned from this research will inform combination treatment strategies that improve the efficacy of ADI-PEG20 and extend survival of patients with SCLC and other ASS1-low tumors. The proposed research will provide unique opportunities for the applicant to gain expertise in cancer biology, cancer metabolism, and computational analysis of -omics data—three major goals of the applicant’s training plan. The proposed research will occur over three years of training at Huntsman Cancer Institute and the University of Utah, a collaborative and resource-rich training environment, in the lab of Dr. Trudy Oliver.