Duke University

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY It is now widely recognized that the cGAS-STING pathway plays a critical role in determining the efficacy of radiotherapy. Radiation-induced DNA damage can cause the leakage of nuclear dsDNA fragments into the cytoplasm, activating the cGAS-STING pathway and inducing type I interferons and antitumor immune response. However, the radiation-induced antitumor immune response is self-limiting, with many molecular mechanisms negatively regulating it. Therefore, a better understanding of the suppressive molecular mechanisms is necessary to enhance the beneficial effects of the radiation-induced antitumor immune response. In this project, we intend to study the role of PCSK9, a critical cholesterol-regulating factor, for its role in regulating the antitumor immune response. We base our project on our recently published study that reveals PCSK9’s role in binding and promoting the degradation of MHC class I on the surface of tumor cells, thereby limiting intratumoral T cell activation (Liu et al., Nature, 2020, PMC7770056). We also base our project on our unpublished preliminary data suggesting that inhibiting PCSK9 can significantly enhance the antitumor efficacy of radiotherapy. We will use genetic approaches to study the roles of PCSK9 in regulating tumor growth and the tumor immune microenvironment after radiotherapy in both transplanted and genetically induced autochthonous murine tumor models (Specific Aim 1). We will also use genetic approaches to determine how PCSK9 interacts with the cGAS- STING signaling pathway in regulating tumor growth and the tumor microenvironment after radiotherapy, with a particular emphasis on STK11/LKB1-mutant tumors, which are known to have a suppressed cGAS-STING signaling (Specific Aim 2). Finally, we will also determine if using a clinically approved PCSK9 inhibitor evolocumab can enhance radiotherapy and immune checkpoint blockade (ICB) therapy of locoregional and distant tumors. We expect our results will inform future PCSK9 inhibitor-based radiotherapy and immunotherapy clinical trials upon completing our project.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Gliomas, including oligodendroglioma and astrocytoma subtypes, are a diverse group of malignant primary brain tumors that respond to radiation, surgery and chemotherapy; however, relapse remains a major barrier affecting overall patient survival. Immunotherapy targeting the adaptive immune system such as checkpoint inhibitors has shown limited efficacy in gliomas. Thus, understanding the immunobiology of gliomas and mechanisms of resistance to immune therapies is crucial to therapeutically leverage the immune system for treating patients. Our long-term goal is to dissect the innate immune system in gliomas and identify vulnerabilities that can be exploited for designing therapies. Recent studies have implicated a link between mutations in ATRX, a SWI-SNF chromatin remodeler and immune cell infiltration in the tumor microenvironment of ATRX-mutant astrocytomas. Our preliminary data suggest that ATRX inactivation in gliomas leads to enriched inflammatory signatures and potentiation of type I interferon/pro-inflammatory signaling, and selective sensitization of tumors to double-stranded (dsRNA)-based immune agonists. Based on these preliminary findings, we hypothesize that ATRX inactivation induces innate inflammation and sensitizes tumors to immune surveillance and dsRNA agonist therapy; concurrent IDH mutations suppress innate inflammation to enable tumor immune evasion. We will test our hypothesis in the following specific aims. Aim 1: Define the role of ATRX inactivation in modulating glioma cell-intrinsic innate signaling; Aim 2: Elucidate the role of ATRX deficiency and concurrent IDH1R132H mutation in modulating anti- tumor immunity and the response to dsRNA agonist therapy in pre-clinical murine glioma models; Aim 3: Determine the extent to which dsRNA-based therapies induce inflammatory activation of lower-grade gliomas. Our proposal will: 1) delineate the novel role of ATRX loss in regulating innate immune signaling responses and their downstream effects in glioma, 2) examine the immunological interplay between ATRX mutations and its partner mutation, IDH1R132H and 3) lay preclinical groundwork for exploiting a potential therapeutic vulnerability in gliomas carrying ATRX mutations.

NIH Research Projects · FY 2025 · 2022-08

Multiple Myeloma (MM) is a plasma cell disorder that accounts for ~10% of all hematologic malignancies. Due to high production of IgG in endoplasmic reticulum (ER), MM cells continuously undergo ER stress which is considered an “Achille’s heel” of the disease. This feature makes MM susceptible to the agents that exacerbate ER stress, such as proteasome inhibitor bortezomib. Yet, currently MM is incurable for most patients due to rapidly emerging resistance to proteasome inhibitors. Therefore, identification of novel anti-MM drugs and targets is of high importance. Conversely, an increase in protein export from ER is a part of the adaptive response to ER stress. In the current application, we propose a novel clinically relevant pathway controlling ER homeostasis and resistance to bortezomib in MM via modulation of sphingolipid composition of the ER membrane. Our preliminary data suggest that such modulation affects ER-to-Golgi transport, ER homeostasis and ultimately MM cell viability. Furthermore, we identified 3-hydroxyacyl-CoA dehydratases (HACD3), an enzyme involved in the biosynthesis of very long fatty acids (VLCFA), as an important regulator of ER-to-Golgi export and ER homeostasis. Importantly, HACD3 mRNA levels were increased during MM progression and in MM cells from MM patients refractory to bortezomib-containing therapy. Therefore, in Specific Aim 1, we will functionally characterize mechanisms underlying VLCFA-dependent regulation of ER homeostasis and characterize enzymes upstream and downstream of HACD3 responsible for such regulation. In Specific Aim 2, we will identify mechanisms regulating HACD3 mRNA expression in MM cells. In Specific Aim 3, we will evaluate the efficacy of pharmacological suppression of VLCFAs in MM mouse models.

NIH Research Projects · FY 2025 · 2022-08

Adverse childhood experiences (ACEs) have been linked to increased risk of tobacco use and other substance use disorders. In particular, individuals with a higher number of ACEs are more likely to smoke cigarettes, initiate smoking at earlier ages, progress to heavier smoking, have higher levels of dependence, and are less likely to quit. However, very few human laboratory studies have been conducted to examine interactions between ACEs and risk for smoking, and the mechanisms underlying these associations are poorly understood. Based on several converging lines of evidence, we propose a translational framework in which ACEs are associated with alterations in corticostriatal circuitry contributing to dysregulated reward processing, which in turn increases sensitivity to reinforcing effects of drugs of abuse, including nicotine. The proposed research will apply a laboratory model of initial nicotine exposure using nasal spray to examine subjective reactions and reinforcing effects of nicotine among young adult non-smokers (n=150) with a history of ACEs ranging from 0 to 4 or more. Participants will first complete a functional neuroimaging protocol designed to assess mesolimbic reactivity to monetary reward, prefrontal inhibitory control, and corticostriatal functional connectivity. Subjective reactions to 0, .5, or 1 mg doses of nicotine nasal spray will be assessed during three separate fixed-dose visits. We will then evaluate reinforcing effects of nicotine during a choice session. In general, we hypothesize that increased exposure to ACEs will be associated with greater positive subjective and reinforcing effects of nicotine, that deficits in corticostriatal circuitry will mediate the association between ACEs and nicotine reactions, and that this association will be stronger among women. These results will provide a critical translation from animal models demonstrating consequences of early life stress on neurobiological pathways relevant to addiction. Moreover, this work will help to explain the increased risk for smoking among individuals exposed to ACEs and will have implications for prevention and treatment of smoking in this high-risk population and beyond.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract NPR1 (NONEXPRESSOR OF PATHOGENSIS-RELATED GENES 1) is a master immune regulator in plants. It orchestrates systemic acquired resistance (SAR) by activating PATHOGENESIS-RELATED (PR) genes in response to induction of salicylic acid (SA) during the plant response to pathogenic challenges. Extensive genetic and biochemical studies have shown that NPR1 is a receptor of SA. SA activation of NPR1 triggers genome- wide transcriptional reprograming via NPR1 interactions with a variety of transcription activators and repressors. NPR1 itself is regulated by redox agents, and its proteolytic turnover has profound implications in a wide range of biological functions, including circadian rhythm and resistance to proteotoxic stress in the endoplasmic reticulum. Despite the essential role of NPR1 in plant biology, the molecular details underlying the myriad of NPR1 functions have remained largely unknown. Building upon our initial success in elucidating the structure of apo NPR1 using single-particle cryoEM, here we propose to elucidate the molecular basis of the NPR1 function in plants. Specifically, we will address (1) how SA activates NPR1, (2) how NPR1 interacts with transcription factors and regulators, and (3) how NPR1 is degraded. Information gained from these studies will provide the much-needed mechanistic insights into the SA-NPR1 signaling cascade in plants and help develop disease- resistant crops. As SA is the principle metabolite of aspirin, such knowledge may also contribute to a better understanding of the well-documented medicinal benefits of SA and analogs in humans.

NIH Research Projects · FY 2026 · 2022-08

Nucleic acids are poised to become the next generation of cancer therapeutics, and the current standard for nucleic acid delivery is lipid nanoparticles. Lipid nanoparticles can effectively deliver mRNA and siRNA in vaccine and therapeutic settings, both preclinically and clinically. This work seeks to develop a polymer-based delivery system for nucleic acids, which may hold multiple advantages over current lipid nanoparticle technologies. A Poly(2-0xazoline) platform for delivering mRNA can produce smaller particles sizes and utilize PEG alternatives in forming polymer-nucleic acid complexes. Smaller sized particles can reduce the significant passive accumulation in the liver and non-PEG polymers can mitigate anti-PEG antibody-based responses. This proposal will investigate these particles in the setting of triple negative breast cancer. Triple negative breast cancer is heterogeneous and highly immunosuppressive, with poor infiltration of key immune cells rendering modern therapies ineffective. By increasing the presence of these key immune cells, therapeutic outcomes can be improved in triple negative breast cancer. This work proposes to deliver mRNA for the key chemokine, CXCL 1, to recruit conventional type 1 dendritic cells to the tumor. These cells are responsible for identifying tumor antigens, translocating to the lymph node, and training T-cells to identify and combat cancer cells. With increased T-cell infiltration, tumors will increase in their sensitivity to checkpoint inhibitor therapy, a critical piece of current treatment paradigms. Our polymer-nucleic acid complexes will target cancer associated fibroblasts, the most abundant cell in the tumor microenvironment, for CXCL 1 mRNA delivery. Aptamers targeting cancer associated fibroblasts will allow us to improve mRNA delivery efficiency to the solid tumor, while mitigating potential side effects from off-target delivery. By hijacking cancer associated fibroblasts, we can utilize a cell which is a key regulator of the immunosuppressive microenvironment to attract dendritic cells to the tumor for antigen presentation-making them a key contributor to the anti-cancer response. Dendritic cells will go on to present antigen to T-cells, priming them for anti-cancer activity and homing to the tumor. This will sensitize these tumors to checkpoint inhibitor therapy by increasing the number of T-cells in the tumor. The successful completion of this project will validate the use of targeted polymer-nucleic acid complexes for gene delivery and demonstrate the viability of chemokine-based treatment paradigms. This is the first step towards chemokine-cocktail immunotherapies, which could target multiple immune cell populations for precision activation of the immune system against cancer.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer that can arise de novo, but more commonly develops after hormone therapies for advanced prostate adenocarcinoma (PADC). It accounts for up to 25% of deaths related to prostate cancer. Current treatment options for NEPC are only palliative, and most patients die within several months. Therefore, there is a pressing unmet need to develop effective targeted therapies for patients with NEPC. Among molecular events associated with NEPC, loss of retinoblastoma (RB) protein occurs nearly universally and drives prostate cancer castration resistance, metastasis, lineage plasticity, and lethality, which suggests that RB1 loss is a pivotal event in the development of NEPC and may be exploited to identify and target therapeutic vulnerabilities in NEPC. In our recent research into the molecular and genetic events underlying ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, we discovered that RB1 disruptions significantly sensitize prostate cancer cells to ferroptosis, at least in part, through a RB/E2F/ACSL4 axis, and that ferroptosis inducers preferentially kill RB1-null NEPC cells rather than RB1-intact PADC cells, implying the therapeutic potential of ferroptosis inducers in the treatment of NEPC. Given that NEPC is notoriously hard to treat and monotherapy often benefits only a small portion of patients, as is the cases with other poorly differentiated neuroendocrine tumors such as small-cell carcinoma of the lung, we propose to develop an effective combinatorial therapy for NEPC based on targeting ferroptosis. Our exciting unpublished preliminary data has shown that the combination of the ferroptosis inducer with the BCL2 inhibitor strongly induces synergistic cytotoxicity in NEPC cells both in vitro and in cell line-derived xenograft (CDX) models of NEPC. Based on these compelling preliminary findings, we hypothesize that ferroptosis inducers and BCL2 inhibitors synergistically promote cell death pathways in NEPC cells, and that co-targeting ferroptosis and BCL2 represents a promising combinatorial approach to treating lethal NEPC. Through a multidisciplinary approach combining unique prostate cancer model systems, in vivo preclinical studies, and well-established molecular and cellular assays, we aim to determine whether co-targeting ferroptosis and BCL2 represents a promising combinatorial approach to treating lethal NEPC. In Aim 1, we will determine the therapeutic efficacy of ferroptosis induction combined with BCL2 inhibition in patient-derived xenograft models of NEPC. In Aim 2, we will determine the therapeutic efficacy of ferroptosis induction combined with BCL2 inhibition in genetically engineered mouse models of NEPC. In Aim 3, we will elucidate the molecular mechanisms underlying the anti-tumor activity of ferroptosis induction combined with BCL2 inhibition in NEPC.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Despite the dramatic improvement in HIV-associated morbidity and mortality with combination antiretroviral therapy (ART), HIV remains a chronic disease. The major barrier to HIV cure is the long-term persistence of multiple, latent viral reservoirs capable of reactivation in the absence of ART. Any effort to eradicate these reservoirs as part of a cure initiative requires understanding of the dynamics and control of HIV reactivation and replication in tissues and cells harboring the virus long-term. Our work has focused on understanding the mechanisms and implications of HIV infection of the kidney. We demonstrated that HIV infects renal tubule epithelial cells (RTEs) in vitro via direct contact with HIV-infected T cells and macrophages. Viral nucleic acid sequence analysis from in vivo derived RTEs compared to blood derived sequences demonstrated that the kidney represents a unique viral compartment. Furthermore, we showed that people with HIV (PWH) shed viral RNA in urine, and we have optimized approaches to detect and amplify HIV sequences from fresh and archived urine specimens. We found that some urine-derived HIV sequences were closely related to HIV sequences amplified from RTEs, supporting those cells as one of the sources of urine viruses. Viral detection in the urine allows for repeated sampling of the kidney compartment, which can be particularly useful in viral rebound studies. Additionally, in all of the PLWH we have analyzed so far, we amplified several identical HIV-1 sequences in urine, raising the possibility of clonal expansion of infected renal cells. Indeed, we recently reported that proliferation is one of the cellular fates observed in both actively and latently infected RTEs in vitro, together with hypertrophy and cell-death. Whether proliferation of infected renal epithelial cells contributes to HIV persistence in the kidney is unknown. The studies proposed here will define: 1) the long-term persistence of HIV in the kidney through the analysis of samples collected prospectively from PWH undergoing HIV+ to HIV+ kidney transplantation; 2) the reactivation potential of HIV in urine following ART interruption in terminally ill PWH who have consented to prospective follow-up as part of a rapid autopsy protocol; 3) the ability of patient-derived renal epithelial cells to carry replication competent virus; 4) the role of APOL1 kidney disease risk variants in RTE and podocyte infection; and 5) how HIV infection influences individual cell fate and potential for clonal expansion of infected RTEs. We hypothesize that renal epithelial cells serve as a long-term reservoir for HIV. Understanding the mechanisms of HIV persistence and reactivation in the kidney will inform cure strategies and further define renal pathogenesis in PLWH.

NIH Research Projects · FY 2026 · 2022-07

Project Summary/Abstract Transgender people experience economic and psychosocial inequities that make them particularly vulnerable to COVID-19 pandemic-related financial and mental health harms. Sustainable, multilevel interventions are needed to address these harms and promote COVID- 19 prevention behaviors. Transgender-led organizations have been galvanized to provide emergency financial and peer support for transgender people negatively impacted by COVID- 19. However, the efficacy of these interventions have not been evaluated. Leveraging existing community partnerships and ongoing cohorts, the study seeks to assess the efficacy of feasible, acceptable, community-derived interventions to reduce economic and psychological harms experienced by transgender people in the wake of COVID-19. The specific aims of the project are to (1) compare the efficacy of microgrants with or without peer mentoring to reduce psychological distress and increase COVID-19 prevention behaviors; (2) examine mechanisms by which microgrants with or without peer mentoring may impact psychological distress; and (3) explore transgender participants' intervention experiences and perceived efficacy. These aims will be met by enrolling 360 transgender adults into an embedded, mixed methods, 3-arm, 12- month randomized controlled trial. Participants will be randomized 1:1:1 to the following arms: (a) a single microgrant plus monthly financial literacy education (usual care); (b) usual care plus monthly microgrants; or (c) usual care plus monthly microgrants combined with peer mentoring. All intervention arms will last for 6 months, and participants will complete semi-annual web- based surveys at 0, 6, and 12 months as well as text-based process measures at 3 and 6 months to meet Aims 1 and 2. A subset of 36 participants, 12 per arm, will complete longitudinal in depth interviews at 3 and 9 months to meet Aim 3. In addition to addressing the pressing impacts of the COVID-19 pandemic on a vulnerable health disparities population, this study will advance the science of minority stress and mental health inequities by testing interventions that operate on general stressors – i.e., material hardship and community connection – rather than minority stressors such as enacted stigma. This national, online study will address multilevel – structural and community – factors driving COVID-19 pandemic harms. Its equitable community partnership will ensure that study findings are actionable and disseminated rapidly to inform sustainable community-based responses to the COVID-19 pandemic as well as future emergencies.

NIH Research Projects · FY 2024 · 2022-07

ABSTRACT Women living with HIV (WLWH) are at increased biologic risk for infection with human papillomavirus (HPV) and development of pre-invasive and invasive cervical cancer. The World Health Organization has recently called for HIV-programs to bolster their efforts to prevent cervical cancer through integrated screening services. Understanding and addressing stigma, including the intersection of HIV, HPV and cervical cancer-related stigma, will be crucial to designing interventions that will facilitate the uptake of cancer screening among WLWH and women in high HIV prevalence settings like western Kenya. Cancer-related stigma negatively influences several determinants of cancer screening uptake, including perception of cancer risk, cancer screening benefit, and acceptability of screening methods. In our prior work to evaluate HPV-based screening in Kenya, we found that lack of education about cervical cancer and low awareness of screening benefits, both of which can potentiate cancer-related stigma, were major barriers to screening uptake. Our team also found that misperceptions and stigma about an HPV diagnosis and cancer were associated with reduced rates of follow-up among women who tested positive for HPV. Our team developed a stigma framework to inform and validate a measurement tool for HPV-, cervical cancer- and HIV-related stigmas. We found that educational messages focused on cancer-related outcomes and HPV epidemiology, including risks related to sexual behavior and HIV, were stigmatizing, while support from social networks and emphasis on the availability of effective treatment reduced stigma and promoted screening uptake. We used this data to develop a stigma-responsive educational intervention which includes simplified scripts for multiple cadres of health workers that provide clear messages about HPV and the benefits of screening and a video aimed at addressing fears and misperceptions from a peer perspective. We propose to incorporate these educational components into “Elimisha HPV,” a multilevel stigma-responsive cervical cancer prevention service delivery model for integration within clinics providing HIV-care in western Kenya. Elimisha HPV, which in Kiswahili means to increase understanding of HPV, will include the following components: HPV-testing via self-collection, simplified scripts and video, peer navigators for women with screening or treatment hesitancy, and the option to receive results and information via text messages. To adapt, implement and test the effectiveness of this model, we will: 1) work with key stakeholders to finalize Elimisha HPV 2) compare cervical cancer prevention outcomes and engagement in HIV care in clinics offering the Elimisha HPV model to clinics providing standard of care outreach, education and screening strategies; and 3) identify individual and institutional factors that moderate the effects of Elimisha HPV on cervical cancer prevention outcomes. If effective, this may represent a new model for HPV-based cervical cancer screening as well as a new paradigm for comprehensive, stigma-responsive service delivery packages for people living with HIV.

NIH Research Projects · FY 2026 · 2022-07

Abstract Despite advances in many domains, the field of solid organ transplantation remains limited by two distinct but connected problems: (1) a critical shortage of donor organs and (2) suboptimal graft longevity due to chronic alloimmune-mediated injury. For patients with end-stage renal disease, these limitations are readily apparent, with over 90,000 individuals in the United States awaiting kidney transplantation. This severe shortfall of donor kidneys is compounded by the suboptimal longevity of transplanted allografts, with a median kidney graft survival of only 8-12 years despite advances in immunosuppression. These significant limitations indicate a clear unmet need to develop novel approaches to improve the function of donor kidneys and enhance graft longevity. The treatment of donor organs with gene therapies has long been recognized as a promising strategy to enhance graft function and diminish graft immunogenicity, but until recently there have not been feasible approaches for gene delivery in an organ-specific manner. Over the last decade, the clinical development of ex vivo organ perfusion systems has created an ideal platform for selectively delivering gene therapies directly to donor allografts. Advancing this approach toward clinical use requires testing in a non-human primate transplant model using clinically relevant immunosuppression regimens. For this proposal, we have assembled a team of investigators with expertise in ex vivo organ perfusion, the use of adeno-associated viral (AAV) vectors for gene therapy, immune management, and kidney transplantation. We have 3 specific aims: 1) Optimize ex vivo machine perfusion approaches for delivery of gene therapies to kidney grafts in an auto-transplant model, 2) Determine the impact of the alloimmune response on transgene expression in kidney allografts, and 3) Evolve novel AAV vectors with tropism for human kidney grafts. Successful completion of this project will demonstrate the use of genetic engineering approaches to achieve durable transgene expression in kidney grafts. This approach has the potential to establish a new paradigm of genetically augmented solid organ allografts and transform approaches in solid organ transplantation.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY While radiation therapy effectively eliminates malignant cells, damage to healthy tissue surrounding tumors remains a persistent clinical issue. Indeed, cancer patients who receive radiation treatment have an increased risk for fracture when compared to those who undergo the same treatment regimen but who are not subjected to radiotherapy. Unfortunately, the use of antiresorptive agents such as bisphosphates does not significantly reduce insufficiency fractures for this patient population. For these reasons, our long-term goal is to identify unique cellular and molecular mechanisms that can be therapeutically exploited for the radioprotection of bone. Notably, the bone microenvironment (BME) is characterized by low oxygen tension or hypoxia. In response to this external stimulus, many cell types in the BME activate hypoxia inducible factor (HIF) signaling to facilitate cell survival. While activation of the HIF signaling pathway is required to maintain healthy bone, the contribution of hypoxia/HIF signaling during radiation induced bone damage has not been well defined. Intriguingly, we show irradiated bones show a decrease in multipotent mesenchymal progenitors (MMPs) when compared to non- irradiated controls. Moreover, preliminary data shows that MMPs are found in hypoxic regions and respond to hypoxia by stabilizing HIF-2. Strikingly, while conditional ablation of HIF-2 in a population of MMPs did not alter bone homeostasis, it did serve to protect against bone loss after radiation exposure. For these reasons, our overarching hypothesis is that genetic and pharmacological inhibition of HIF-2 will serve as a radioprotective mechanism to ameliorate bone damage after radiation exposure, in part, by maintaining the number of MMPs that can functionally contribute to bone after stress induced damage. To test our hypothesis, we will utilize a combination of genetically engineered mouse models, in vitro cell culture experiments, and novel pharmacological approaches to inhibit the HIF-2 signaling pathway in the BME. Currently, there are no FDA approved agents to mitigate radiation induced bone loss, hence these studies will not only expand our fundamental knowledge of bone biology but will also fill an unmet clinical need to identify therapeutic targets which will ameliorate bone damage after radiotherapy.

NIH Research Projects · FY 2026 · 2022-07

Despite experiencing a higher prevalence of depressive symptoms, Latino/a adolescents are significantly less likely to receive treatment for Major Depressive Disorder (MDD) and are more likely to drop out of psychotherapy prematurely than their non-Latino/a White peers. Research is needed to develop patient- and family-focused implementation strategies to promote Latino/a adolescents’ initiation of and retention in psychotherapy for depression. The candidate aims to identify, refine, and test a patient- and family-focused implementation strategy to target obstacles to psychotherapy attendance among Latino/a youth. In Aim 1, a national sample of Latino adolescents (n=15), parents (n=15), and healthcare providers (n=5) will participate in focus groups to identify preferred strategies to promote psychotherapy attendance. In Aim 2, a series of design thinking workshops will be carried out with a community advisory board (N=8) to refine and develop the content of the implementation strategy. Qualitative descriptive methods and rapid content analysis will be employed in aims 1 and 2. In Aim 3, the candidate will conduct a clinical trial to determine the preliminary effect of the patient- and family-focused implementation strategy on psychotherapy attendance and depressive symptoms among Latino/a adolescents (N=60) and identify facilitators and early implementation outcomes (acceptability, adoption, feasibility, and fidelity) related to the implementation of the strategy and psychotherapy among Latino/a adolescents. Latino/a adolescents diagnosed with MDD or Persistent Depressive Disorder and referred to psychotherapy will be recruited from primary care clinics in North Carolina and randomly assigned to the implementation strategy or treatment as usual group (n=30 each). Measurements will be taken at baseline, 12 weeks, and 24 weeks. Wilcoxon Two-Sample Tests will be used to compare group differences in the percent of scheduled psychotherapy visits attended at 24 weeks. Wilcoxon Signed Rank Tests will be used to determine change in the depressive symptoms from baseline to week 12, and baseline to week 24. Qualitative interviews with Latino/a adolescents, parents, and healthcare providers will be conducted to complete a multi-method evaluation of implementation process outcomes. The results of the study will lay a foundation for a full-scale R01 randomized controlled trial. The identified patient- and family-focused implementation strategy might also serve as a model for addressing behavioral health disparities among other groups of youth. During this award, the candidate will also obtain critical training in implementation science, adaptation of programs for Latino/a families, adolescent depression treatment, and clinical trial design. The proposed award will support the candidate’s long-term goal of promoting mental health among Latino/a adolescents and her transition into an independent, health disparities researcher

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract The 1918 influenza pandemic is estimated to have killed 1 in 20 people worldwide. Influenza A virus (IAV) infections usually do not cause such severe disease for the ~30 million infected every year in the United States alone (2014-2015). However, there are broad differences in IAV susceptibility and severity, with outcomes from asymptomatic infections (~16%) to death (0.2% in 2014-2015). These differences arise from the complex interplay of exposure, environment, IAV genetics, and host factors. A crucial host factor that contributes to heterogeneity of IAV infection is sex. For children and older individuals, males are more likely to experience severe disease, while females of child-bearing age have greater severity. As there is strong evidence for 1) the importance of sex in IAV infection, 2) gene expression differences between males and females, and 3) human genetic variation impacting infectious disease in general and specifically IAV infection, synthesis of these three areas may provide crucial mechanistic insight. We hypothesize that sex differences in gene expression are a major driver of heterogeneity in IAV infection. To elucidate these differences, this project will integrate cutting-edge approaches to identify sex-specific differences in transcript abundance and splicing that regulate IAV burden and host response in human cells, IAV challenge volunteers, and natural populations. Further, we will define the genotype x sex interactions that form the mechanistic basis for how genetic diversity contributes to sex differences in IAV infection. To achieve these goals, we have unique datasets of IAV infection heterogeneity in cells from dozens of male and female donors, in nasal curettage and peripheral blood from human IAV challenge subjects, and biobanked samples of natural IAV infection with outcomes ranging from mild infection to death. Computational analyses of these datasets will define 1) sex differences in gene expression that correlate with IAV burden and symptom severity and 2) human SNPs that regulate sex-biased gene expression and flu severity. The transcriptional profiles from these datasets will be used to generate sex-specific biomarkers of IAV infection severity using machine learning approaches. Finally, we will experimentally determine whether the identified sex-biased genes and SNPs regulate IAV burden and host response in cellular models of infection. All results will be available through an easy-to-use web database for exploring this rich dataset as a launchpad for further mechanistic and clinical studies. This project will develop and apply computational methods to generate a high-resolution analysis of how sex and genes interact to impact IAV infection. Understanding the genetic basis for sex differences in IAV infection could lead to new diagnostic approaches in identifying at-risk individuals and novel therapeutic strategies.

NIH Research Projects · FY 2025 · 2022-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Duke Medical Scientist Training Program (MSTP) was originally funded by NIGMS in 1966, and it has been continuously funded since that time. This application requests funding for 24 trainees. The goal of the Duke MSTP is to prepare young physician-scientists for careers in biomedical research and academic medicine by providing comprehensive training in both scientific research and clinical medicine through a highly integrated curriculum that leads to completion of both the MD and PhD degrees. The program's mission is to provide an intellectual foundation that will allow trainees to become the future thought leaders in biomedical science and academic medicine in the US. To accomplish these goals, the Program is built on a pioneering medical school curriculum that integrates medical education with original scholarly investigation. The defining feature of the Duke curriculum is that all medical students engage in significant scholarly activity in the third year. As a consequence, the core medical education takes place over three years, rather than four, with the preclinical basic sciences condensed into the first year and core clinical clerkships taught in the second year. This curriculum is therefore ideally suited to MD-PhD trainees, as it provides a comprehensive clinical foundation for PhD training, allows students to align their research interests with their long-term clinical interests, and shortens the time to the dual degree. This application builds on the Duke MSTP’s long history of training successful physician-scientists by incorporating a number of major changes and improvements, including: 1) Incorporation of improved medical school and graduate school assessment tools to track and evaluate students’ progress throughout their training; 2) Development and incorporation of enhanced opportunities for trainees to influence their own career development; 3) Improvement of the admissions process to ensure selection of outstanding candidates, including enhanced outreach efforts to undergraduate institutions to enhance the pipeline of qualified candidates; 4) Enhanced trainee oversight at multiple stages of the program, including an emphasis on identification of roadblocks during annual Individual Development Plan meetings between leadership and students; 5) Ensuring participation of outstanding faculty at all career levels; and 6) Creation of additional program activities with guidance from the program’s trainees and steering committee to ensure adequate program-wide training opportunities that foster program community, including enhanced vertical integration through Duke’s institution-wide Office of Physician-Scientist Development. Collectively, these new program initiatives will continue the Duke MSTP's longstanding tradition of excellence in MD-PhD training and substantially improve outcomes for the approximately 85-90 students in the program each year.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Rearrangement of NUP98 gene (NUP98-r) is recurrent in leukemias such as acute myeloid leukemia (AML). Patients with NUP98-r show poor prognosis and therapy failure. Most NUP98-r partners (>30 identified from patients) are a DNA-binding domain of transcription factor (TF; e.g. HOXA9) or a histone-binding motif such as Plant Homeodomain (PHD), suggesting chromatin deregulation as an oncogenic mechanism. NUP98-r fusions invariably retain Phenylalanine-Glycine (FG) repeats, termed intrinsically disordered region (IDR), from NUP98. How unstructured IDR contributes to oncogenesis remains elusive. Our studies of NUP98-HOXA9, an AML NUP98-TF chimera, unveil an essential requirement of NUP98’s IDR for liquid-liquid phase separation (LLPS). We also show that IDR and LLPS are critical for the much-enhanced genome binding by NUP98-HOXA9 and for long-distance chromatin looping between oncogene promoters and enhancers, leading to development of aggressive AML in mice. Our unpublished preliminary studies of other recurrent leukemic fusions (namely, NUP98-PHD fusions and MSI2-HOXA9, a leukemia-related chimera formed by fusing a less-studied IDR of an RNA-binding protein with HOXA9’s DNA-binding domain) all point to involvements of IDR and LLPS for oncogenesis. Thus, we hypothesize that, due to aberrant genic fusions, a number of leukemia-related onco- TFs and chromatin factors acquire a phase-separation-inducing IDR to establish LLPS, which confers chimera a much more enhanced ability in genomic targeting; consequently, an oncogenic gene-expression program is over-activated while aberrant chromatin loops are formed between oncogene promoters and enhancers, which drives formation of aggressive leukemias. Dissection of the mechanisms underlying the IDR- and phase- separation-mediated aberrant genome organization and oncogene activation in cancer cells shall provide new and paradigm-shifting views as for how aggressive cancer develops, implicative of potentially new treatments in future. Towards this goal, we will further characterize the role for the un-studied IDR (that of MSI2) in establishing LLPS in vitro and in cells (Aim 1A) and will use primary human hematopoietic stem/progenitor cells (HSPCs) and derived cells to define roles of IDR and LLPS in regulating genomic targeting (1B), the target gene expression (1C), and leukemic transformation in vitro/vivo (1D) by various fusions (NUP98-PHD and MSI2/NUP98-HOXA9). LLPS-indued chromatin looping is CTCF-independent and represents a previously unstudied mechanism underlying 3D chromatin organization. We will further define the 3D chromatin structure alterations caused by various NUP98-r and MSI2-HOXA9 fusions in disease-relevant cells (Aim 2A), define the molecular mechanisms driving formation/maintenance of LLPS-dependent loops (2B), and determine the impact of LLPS DNA loops on the sustained activation of oncogenes by using a novel CRISPR/dCas9-IDR fusion strategy (2C). As phase-separation-competent molecules are frequently implicated in a wide range of human cancers and diseases, both the significance and overall impact of the project are potentially high.

NIH Research Projects · FY 2025 · 2022-07

Diffuse traumatic brain injury (TBI) is associated with various pathologies that lead to long-term impairments, including post-traumatic headache (PTH), particularly migraine. There are worse outcomes of TBI when compounded with elevated intracranial pressure (ICP). The objective of the F99 work is to elucidate the mechanism(s) that may be attributed to TBI-induced pathologies and ICP-mediated diffuse pathologies. The objective of the K00 is to investigate cellular and molecular mechanisms of migraine to become an investigator of PTH in TBI. One pathology, neuronal membrane disruption, has been shown to be induced acutely post-TBI. However, my initial studies present that membrane disruption in neurons can last beyond the acute timeframe and last for weeks post-TBI. Furthermore, data indicate that there is a subpopulation of cortical neurons that do not express NeuN and are membrane disrupted. With other injuries, a NeuN negative (NeuN-) presentation has been indicative of a reversion to an immature neuronal phenotype. If injured neurons are reverting to an immature phenotype, then this could be a compensatory mechanism, so identifying these neurons that also are membrane disrupted may provide more insight into the molecular mechanism of membrane disruption. Aim 1a) is to investigate the identity of NeuN- subpopulation of membrane disrupted neurons using intracerebroventricular (ICV) cell impermeable fluorescently-tagged dextran tracer in sham and central fluid percussion injured (CFPI) animals. Then using histological and molecular paradigms, I will evaluate membrane disrupted neurons for cellular NeuN expression, and expression of immature neuronal markers. Incidentally, the mechanism behind neuronal membrane disruption is unknown, and previous work from our lab shows that when TBI is compounded with an elevation in ICP in rats, that neuronal membrane disruption is increased in direct relation to the ICP elevation. Aim 1b) is to evaluate the effects of secondary ICP elevation on the NeuN- membrane disrupted subpopulation. I propose that lysosomal Cathepsin B (Cath B) is a potential mediator of membrane disruption, as previous findings reveal that Cath B re-localizes from the lysosome to the cytosol, which has been shown by other groups to initiate cell damage/death. I intend to evaluate the effects of secondary ICP elevation on the NeuN- membrane disrupted population using sham, CFPI animals and CFPI+elevated ICP animals with the same dextran protocol aforementioned, via microscopic and molecular approaches. Simultaneously, I will investigate the role of Cath B in vivo by inhibiting Cath B following sham and injury then using activity assays to verify inhibition as well as microscopic studies to evaluate the re-localization of Cath B. I expect in the F99 project that diffuse TBI paired with elevated ICP will see increases in membrane disrupted population later. Yet, a reduction in the compensatory NeuN- subpopulation as these neurons will endure a secondary insult. Finally, it is expected that Cath-B is re-localizing from the lysosome to the cytosol after TBI in membrane disrupted neurons and will be exacerbated with secondary ICP insult.

NIH Research Projects · FY 2025 · 2022-07

STEM identity – the view of oneself as a scientist or engineer – plays a crucial role in students’ motivation to pursue STEM education and careers. Providing pre-college students with project-based experiences that challenge them to employ STEM thinking and practices is essential in STEM identity development. The burden of creating these opportunities falls largely on teachers, who may feel ill-equipped to impart these concepts, especially in teaching engineering and engineering design - an area of emphasis in the widely adopted Next Generation Science Standards. This is unfortunate because engineering design is an exciting entry point for STEM that involves inquiry, project-based learning, and the application of technical skills and knowledge to real-world problems. The Hk Maker Lab has provided engineering design-centric education to hundreds of high school students and has prepared educators to teach engineering design and incorporate design thinking into their classes. We are now ready to extend our efforts to middle school students and teachers to further propel grades 6-12 students toward STEM opportunities at a crucial juncture in their educational journeys. The Specific Aims of the proposed program are to: Provide early engineering education opportunities to middle school students via Design Hackathons that will take place throughout the academic year, in addition to continuing the annual Summer Design Camp for high school students. Expand teacher training to middle school teachers to develop their engineering design knowledge and self-efficacy so that they can integrate design into their instruction. Cultivate a cohort of master ‘Teachineers’ who are highly adept in engineering design thinking and instruction and can teach design thinking to other teachers via professional development workshops. This research will give both teachers and students hands-on design experiences. The student participants will have the opportunity to engineer solutions to problems. Working with middle school students will create a positive STEM trajectory. The participating teachers will receive an innovative training experience, which they can subsequently use to integrate engineering design into their school curricula. Finally, by empowering teachers to train their colleagues, we will be able to scale these efforts, increasing the impact of our engineering design training and broadening STEM education opportunities for all students.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Candidate: Deborah Kaye, MD, MS is a urologic oncologist and junior health services researcher focused on improving the outcomes for patients with cancer. Dr. Kaye’s long-term goal is to become an independent surgeon-scientist with expertise in quantitative and qualitative research methods, an in- depth understanding of provider decision-making and intervention and clinical trial design, in order to devise, evaluate and implement effective health care policies to improve the lives of patients with cancer. Research Context: Numerous systemic therapies have recently been approved for the treatment of advanced prostate cancer, demonstrating modest improvements in median survival fromtwo to five months. Unfortunately, little is known regarding which therapies, or combinations of therapies, are most effective. What is known, however, is that each option differs significantly in route of administration, side effects, and price. Currently, treatment for advanced prostate cancer varies significantly based on provider specialty and location, and patient race and income. With sparse comparative effectiveness evidence, treatment recommendations rely heavily on physician preference and experience, which likely contributes to differential patient outcomes and payment disparities. Economic and non-economic drivers of provider decision-making remain a critical knowledge gap in advanced prostate cancer. Moreover, few interventions exist to decrease the out-of-pocket payments, and therefore potentially improve outcomes for patients with advance prostate cancer. Specific Aims: 1) To determine the factors contributing to provider decision-making for initial choice of systemic therapy for advanced prostate cancer. 2) To develop a cost-transparency intervention to decrease patient OoP payments for advanced prostate cancertreatment. 3) To pilot test the feasibility, acceptability, usability and initial signals of efficacy of a cost-transparency intervention. Research Plan: Dr. Kaye will use qualitative research methods (focus groups, patient and provider interviews) to determine the factors impacting provider decision-making and type of first-line systemic therapy received, and use iterative intervention design to devise a cost-transparency intervention to lower patient out-of-pocket payments and improve outcomes for patients with advanced prostate cancer. Finally, this proposal will test the feasibility, acceptability, usability and initial signals of efficacy of the cost-transparency intervention with a two- arm randomized pilot trial Career Development Plan: In parallel with the outlined research plan and under the guidance of experienced mentors, Dr. Kaye will develop expertise in: 1) provider decision-making; 2) qualitative research methods; 3) intervention and clinical trial design.

NIH Research Projects · FY 2026 · 2022-07

Project Summary/Abstract Dementia is a common disorder that increases in frequency in the elderly. Dementia is a progressive loss of thinking and memory skills that eventually results in an inability to care for oneself and to live independently. The most common cause of dementia in older Americans is Alzheimer’s disease, which develops as a result of multiple lifestyle/environmental factors and inherited or familial causes. Many familial traits (such as the risk of developing Alzheimer’s disease are inherited genetically, through gene variants (sequences of a chemical code called DNA). The most common genetic variant that increases the risk of Alzheimer’s disease in older adults (ie above age 60) is called APOE4. Although we have known for over 25 years that inheriting an APOE4 genetic variant (also called an allele) increases Alzheimer’s disease risk, it is unclear how and why APOE4 increases this risk. Recent data has raised the possibility that APOE4 may act by increasing the activation of the complement pathway, a set of proteins that are used by immune cells to kill and eat bacteria, and that also play a role in “killing” and “eating” connections between nerve cells in the brain, known as synapses. In this project, we will examine whether people that carry the APOE4 allele (either 1 or 2 copies of this allele) have increased evidence of complement pathway activation in the fluid that bathes their brain and spinal cord, known as the cerebrospinal fluid (CSF). We will also determine whether CSF complement pathway activation in APOE4 allele carriers is associated with long term cognitive decline over a 7.5 year period. Finally, we will determine whether modulating APOE biology in vivo with a peptide that “mimics” the disease-resistant APOE2 allele. This work will be performed using an advanced method that can measure the levels of a large percentage of all proteins present in human CSF, and which can precisely measure the levels of proteins in the complement pathway. Overall, this work will help us understand how and why APOE4 changes the function of the brain, and how and why it contributes to AD risk. This work is an early step towards identifying proteins or pathways that could be targeted by drugs to prevent Alzheimer’s disease in people that carry an APOE4 allele.

NIH Research Projects · FY 2025 · 2022-07

An emerging feature of cardiometabolic disease states, including obesity, diabetes, and heart failure is perturbed metabolism and subsequent elevations of plasma branched-chain amino acids (BCAA; valine, leucine, isoleucine) and their cognate α-ketoacids (BCKA; KIV, KIC, KMV). Work from our group and others has revealed that elevated plasma BCKA arise in these conditions due to impaired activity of the branched chain a-ketoacid dehydrogenase (BCKDH) complex in liver, resulting from higher expression of the inhibitory BCKDH kinase, BDK, and lower expression of the activating phosphatase, PPM1K. Importantly, whole-body manipulation of BCKA metabolism achieved via pharmacologic or genetic modulation of BDK and PPM1K yields robust impacts on cardiac structure, function, and metabolism. Thus, novel therapeutic approaches targeting BCKA-related pathways hold significant potential for treatment of cardiac dysfunction. However, it remains unclear whether the in vivo effects of systemic BDK and PPM1K manipulation on cardiac function are due to modulation of PPM1K and BDK activity within the heart or simply the result of chronic exposure of the heart to increased plasma BCKA. The work outlined in this proposal will build upon our prior work to directly address this important knowledge gap by leveraging newly developed animal models to: 1) Determine the impact of liver-specific BDK modulation on cardiac function; 2) Extend our mechanistic understanding of BCKA-mediated signal transduction pathways in the heart; and 3) Define the role of PPM1K in the heart. The knowledge gained from the completion of this project is expected to aid in development of BCKA-related therapeutic targets for the treatment of cardiac dysfunction in a wide range of cardiometabolic diseases.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract Emerging infectious diseases pose a significant risk to human health. Among emerging pathogens, hemorrhagic fever viruses (HFVs) pose the greatest risk in terms of the potential for morbidity and mortality. These viruses are endemic in remote regions, produce large outbreaks that display high mortality rates, and have a high potential become pandemics. One of the largest families of HFVs is the Arenaviruses, which includes 6 DHHS select agents. The most well-known of these, Lassa virus, has caused an increasing number of infections in recent years, including a 2018 outbreak in Nigeria that led to over 100 deaths. In 2017, the World health Organization designated Lassa virus as a priority pathogen for R&D efforts due to its potential to generate a public health emergency. Other hemorrhagic fever arenaviruses, including Lujo, Chapare, Guanarito, Junin, and Machupo represent an increasing source of concern based on their increased distribution and recent instances of human-to-human transmission. Current diagnostic capabilities for Lassa virus and other arenaviruses are extremely limited, especially in the case of point-of-care (POC) diagnostic assays. Here, we propose to develop a POC diagnostic assay with the sensitivity and breadth of coverage required to diagnose Lassa virus infections currently present in West Africa. In addition, we will generate single chain antibody phage display libraries, broadly reactive high affinity diagnostic antibody pairs, a prototype assay chips for the 5 other arenaviruses that are Category A Priority Pathogens. We have previously used these procedures to develop a POC diagnostic assay for Ebola virus that displays a sensitivity better than the current gold standard, PCR. The successful completion of these studies will result in the first POC diagnostic assay that is capable of detecting all Lassa strains of clinical importance and a panel of high affinity diagnostic Abs and assays for all Category A Arenaviruses. In addition, the broadly reactive single chain antibody libraries we produce could be used to rapidly generate diagnostic antibodies against novel arenaviruses that arise as zoonotic outbreaks. This work will thus significantly improve our preparedness for almost any future major Arenavirus outbreak with pandemic potential.

NIH Research Projects · FY 2025 · 2022-07

Abstract. This proposal focuses on the human fungal pathogenic Mucor species complex, a group of related pathogens that cause devastating infections that are difficult to treat, with limited drug treatment options, and requiring surgical debridement in some patients. Over the past decade, we and others have advanced genomics, genetics, and animal models for this understudied group of microbial pathogens. We discovered the protein phosphatase calcineurin controls the dimorphic transition from yeast to hyphae required for Mucor pathogenesis, and through studies of FK506-resistant isolates discovered a novel mechanism of antimicrobial drug resistance. In previously published and preliminary studies, significant advances were achieved through our discovery of a novel mechanism of antifungal drug resistance called epimutation, whereby the RNAi pathway is activated and silences drug target genes. This pathway confers transient, unstable drug resistance, and resistant isolates rapidly revert to drug sensitivity in the absence of drug. Through genetic and molecular studies, we defined RNAi components required for epimutation, those that are dispensable for epimutation, and a novel category that inhibits formation of epimutations. The discovery of antimicrobial drug resistance mediated via epimutations has been generalized: 1) showing epimutation occurs in two different pathogenic Mucor species, 2) defining an alternative RNAi pathway controlling epimutation frequency and stability, 3) identifying epimutations in additional genes causing resistance to antifungal agents, and 4) documenting that epimutations persist during animal infection or arise after animal passage. These insights set the stage for studies proposed here to further define mechanisms of epimutation, and elucidate the impact of epimutations on microbial pathogen interactions with the host. In the current proposal, we hypothesize epimutation is a general process that operates across many eukaryotic microbial pathogens, and acts as a major force in antimicrobial drug resistance that controls target genes involved in drug action, genome stability, and pathogenesis of eukaryotic microbial pathogens. Our studies will reveal unique facets of RNAi that lead to epimutations, which mediate antimicrobial drug resistance in ubiquitous fungal pathogens of humans. Aim 1 will 1) elucidate molecular mechanisms of epimutation and targets, including genes involved in drug resistance (including clinically used antifungal drugs) and transposable elements, and their impact on genome stability, 2) define conditions, including stress, sexual reproduction, and infection, that may drive the emergence of epimutations, and 3) establish the generalizability of these findings to other pathogenic fungal species. Aim 2 will define the impact of epimutation on antimicrobial drug resistance and pathogenicity in microbe interactions with immune cells, the blood-brain barrier, organoids, and whole-animal models. These studies will advance our understanding of how antimicrobial drug resistance can evolve via a novel RNAi-based pathway with direct implications for infectious disease evolution, treatment, and prevention, and provide insights into other eukaryotic pathogens with active RNAi pathways.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Clinical outcomes in invasive gram-negative bacterial infections are determined by the interplay of patient, treatment, and bacterial variables. Significant progress has been made in understanding patient and treatment factors; however, little is known about how bacterial genetic variation influences patient outcomes. This is an important gap in understanding microbial pathogenesis and limits the ability to identify drug targets critical in human pathogenesis. To address this problem, the long-term goal is to interrogate bacterial pathogenesis in humans by defining the impact of bacterial genetic variation on the outcome of patients with gram-negative bacteremia. This knowledge can be leveraged to identify novel treatments. The career development plan reflects this long-term goal as the emphasis is to develop quantitative and lab expertise to independently identify and explore associations between bacterial genetics and patient outcomes. The objective in this proposal is to characterize two putative pathogenicity islands (PPI), variably present in E. coli, that are promising for their roles in pathogenesis and potential as drug targets. The two PPI were identified by the applicant and encode genes homologous to a type III secretion system (T3SS) structural apparatus (PPI-1) and translocases/adhesin (PPI-2). PPI-1/PPI-2 are not present in typical laboratory strains of E. coli and so have not been significantly studied. Presence of PPI-1/PPI-2 associated with increased mortality in patients with E. coli bacteremia, and deletions in PPI-1/PPI-2 allowed for complement-mediated killing of E. coli in serum and impaired E. coli-host cell interactions (decreased invasion). Further, PPI-1/PPI-2 functioned as a prognostic biomarker that improved ability to identify patients at high risk of mortality. The central hypothesis is that PPI-1 and PPI-2 function together as a T3SS that mediates complement-mediated serum resistance and tissue invasiveness to influence patient outcomes, and presence of PPI-1/PPI-2 provides prognostic value in patients with E. coli BSI. This will be tested through two specific aims (SA): 1) Determine how PPI-1 and PPI-2 affect virulence to influence patient outcome, and 2) Define value of PPI-1 and PPI-2 genotype as prognostic biomarkers for E. coli bacteremia. SA1 will identify how PPI-1/PPI-2 mediates resistance to complement activation, verify that PPI-1/PPI-2 is a functional T3SS, and determine the relative contributions of serum killing versus T3SS function to virulence. SA2 will use an existing external validation cohort to show that incorporating bacterial genetics with clinical variables improves prognostic models of clinical outcomes in E. coli bacteremia. Characterization of PPI-1/PPI-2 will increase our understanding of E. coli pathogenesis and pave the way for novel therapeutics. For example, a protein in PPI-2 is homologous to a drug target that the applicant and others previously exploited in Pseudomonas aeruginosa. The skills and mentoring acquired during this award will scaffold development into an independent clinician-scientist focused on the critical area of gram-negative infections.

NIH Research Projects · FY 2025 · 2022-07

The current proposal seeks to continue support of the long-standing biotechnology training program administered by the Center for Biomolecular and Tissue Engineering (CBTE) at Duke University. The objective of this biotechnology training program is to provide predoctoral training in the classroom, laboratory, and scientific community in the design, manipulation, and quantitative characterization of biomolecules, cells and tissues with special emphasis on the translation of these technologies and their advancement within the biotechnology industry. Training activities are focused on enhancing and elevating our biotechnology work force and preparing the next generation of leaders who will pioneer burgeoning new fields such as biologic drug development, tissue engineering, and gene and cell therapy. Participating predoctoral trainees are required to meet the following criteria: (1) perform research that is interdisciplinary in nature and is central to the development of innovative biotechnology; (2) have at least two CBTE faculty, one from biomedical sciences and one from engineering, on their doctoral dissertation committee; (3) present in the monthly work-in-progress student seminar series; (4) complete engineering and biomedical science courses that provide breadth in biomolecular and tissue engineering; (5) complete four semesters of the interdisciplinary CBTE seminar series for credit, including dedicated meetings with visiting speakers; (6) participate in a three-month industrial biotechnology internship; (7) present in the annual CBTE Distinguished Lecture and Symposium; (8) attend regularly scheduled biotechnology-focused career development seminars and workshops; and (9) undergo training in responsible conduct and rigor and reproducibility in research. This interdisciplinary training program includes 37 faculty across campus, with 22 faculty from the Pratt School of Engineering and 15 faculty from non-engineering fields, including 5 in the Trinity College of Arts & Sciences, and 10 in the Basic Medical Science and Clinical Departments of the Duke University Medical Center. Over the past 26 years, a total of 116 students have received predoctoral traineeships in biomolecular and tissue engineering. Most of our trainees have obtained positions as leaders in the biotech industry, including as co-founders of new companies and pioneers in new areas of biotechnology, while others have pursued careers in academic, medicine, or government. Collectively, these activities decidedly increase the value of the education and preparation of our trainees for careers as leaders in the biotechnology industry and other fields, as demonstrated by our extensive track record and quantitative outcome measures.