University Of Wisconsin-Madison

NIH Research Projects · FY 2024 · 2022-03

ABSTRACT Optical imaging of Cherenkov emission from tissue during radiotherapy has been demonstrated in humans, providing a map correlated to radiation dose within the surface tissues. In external beam radiotherapy, the signal is optimally captured by time-gated intensified cameras, synchronized to the linear accelerator pulses, allowing rejection of the majority of background room light, and providing real time video of each radiotherapy treatment with standard dose rates. This discovery combined with CT and reflectance calibration has the potential to allow accurate quantitative dose imaging in humans for the first time in the history of therapeutic radiation use. While the imaging technology has inherent limitations to surface regions, the fact that it is a real time imaging tool and provide visual recording of every feature of a treatment plan is revolutionary. It has the potential for a paradigm change in how radiotherapy is documented and archived for daily reliance, quality audit and potentially automated control of delivery. The implementation of Cherenkov imaging is significantly simpler than most dosimetry tools, however the understanding and interpretation of the images need to be established, in order to be clear about what is possible in imaging delivery of dose. This proposed technology developments advance the methods for quantitative dose imaging in whole breast radiation treatment using our patented approach to CT/reflectance calibration of Cherenkov imaging, will be advanced and tested in regular treatments. Additionally, we will advance the ability to image patients with complex multi-field treatments, where adjacent beam fields are matched, so that we can quantify the dose accuracy at the match lines. Another application is treatment of total skin with electron therapy, where the field is extremely large and so imaging dosimetry makes sense. Our preliminary data indicates that areas of chronic underexposure exist today and the optimal delivery technique will be defined by our efforts in imaging and 3D animation & visualization of total body skin dose, developing an optimized imaging system to identify the ideal treatment technique for patients of different body shape. Finally, Cherenkov imaging is synergistic with scintillator imaging of dose, and this will be advanced as a related tool to remotely quantify skin dose. Taken together, this project will advance the only way to directly image radiotherapy dose and delivery on the patient’s tissue. The core of the project is combined technology systems, leveraging a large installed base of cameras and national partnerships advancing this field. This technology is embryonic but on the cusp of commercialization, where the focus of this research project being to advance the science and methods of where it can add value to radiotherapy. The long term benefit of advancing this technology will be to change the way in which radiotherapy incidents are observed by the therapist team and have these recorded on a daily basis in every treatment center.

NIH Research Projects · FY 2026 · 2022-03

SUMMARY Age is the greatest risk factor for a host of chronic diseases, including cancer, diabetes, cardiovascular disease and neurodegeneration. The mechanistic basis for this shared risk and its continued increase as a function of age is not well understood. Caloric restriction (CR) without malnutrition has been proven to delay aging in diverse species, and in mammals it delays the onset of numerous age-related diseases, increasing healthspan. The Aging and CR in Rhesus Monkeys study at the Wisconsin National Primate Research Center established the efficacy of CR in improving health and survival: CR monkeys live longer, have lower incidence of age-related diseases, are more active, and maintain better glucoregulatory health. Molecular profiling studies suggest that CR induces a major reprogramming of metabolism, with changes in key cellular homeostatic pathways coordinated across transcriptional, proteomic, and post-translation modification regulatory mechanisms. Our limited studies to date have identified novel aspects in CR's mechanisms including lipid metabolism and signaling, and the role of RNA-based regulatory mechanisms including transcript processing and coordination of the CR response through microRNA. The proposed studies have potential to uncover further regulatory mechanisms engaged during aging and CR at the tissue specific level, derive interaction networks within and among tissues to define the molecular details of how CR works, and relate these data to whole animal physiology, health, morbidity, and survival. This unique cohort of monkeys presents an unprecedented opportunity to advance our understanding of aging biology. Although the intervention of CR may not be a reasonable choice for clinical application, the proposed unbiased high-resolution studies are certain to reveal new insights into how aging itself might be targeted clinically. There are three Specific Aims: Aim 1. Determine shared and tissue-specific mechanisms engaged by CR. Aim 2. Determine the life stage-resolved systemic response to CR. Aim 3. Integrate the physiological, systemic, and molecular responses to CR. Our study is designed to define the integrated response to CR within and among tissues and at the whole organism level in primates, and to determine how these CR-engaged mechanisms might coordinate to confer enhanced longevity. Rhesus monkeys are a highly translational model for human aging, in particular with regards to the timing of onset of age-related diseases and disorders and the dynamics of functional decline. Our cohort is derived from a unique study of effective implementation of CR, with physiological data and specimens in hand, along with substantial longitudinal clinical data, health records, and end of life pathology. Integrative analysis of high-density molecular profiles within and among tissues will present a new perspective in aging biology at the systems level, and by linking to clinical outcomes will deliver translational insights for human aging.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Mitochondria are essential for cellular function and organism viability. These organelles are well known for their production of ATP, the primary energy currency of most eukaryotic cells. Less well known are the plethora of other functions these organelles have including production of signaling molecules, regulation of apoptotic signaling cascades, serving as a calcium sink, and also being the primary storage and utilization site of iron in the cell. To serve these diverse functions, mitochondria must be properly localized in all cells; however, this organelle is particularly critical in neurons. Neurons are highly metabolically active, electrically polarized, and can have an enormous volume making regulation of the mitochondrial population particularly challenging. Likely due to the high metabolic demands of this cell, precise control of mitochondrial localization and maintenance of mitochondrial health are essential for neuronal survival. Abnormal mitochondrial localization, health, and function have been linked to many neurodegenerative diseases including Alzheimer’s disease. In Alzheimer’s, defects in mitochondrial calcium load and contacts with the endoplasmic reticulum have both been noted. Additionally, advanced neuroimaging of early-stage patients revealed defects in mitochondrial function, making understanding how mitochondrial function is maintained in neurons paramount to understanding disease biology. While the last several decades have revealed fascinating insights into mitochondrial biology in neurons, we still do not have a thorough understanding of how the population of mitochondria is maintained over the long life of the neuron. Anterograde transport is critical for bringing healthy organelles from the cell body into the long axonal process which can extend a meter from the cell body in humans. Conversely, retrograde transport moves aged or damaged organelles towards to cell body. Once damaged organelles reach the cell body, some undergo targeted degradation. The fate of the bulk of these organelles and the source of healthy mitochondria has not been defined. We have developed an in vivo system to address these long-standing questions in the field. Using zebrafish neurons, we can image mitochondrial localization, health, and transport in vivo in a fully intact neural circuit. We have developed transgenic lines, genetic tools, and imaging approaches to individually label mitochondria to track them and follow their lifetime and biogenesis in neurons. This will allow us to determine the source of healthy mitochondria necessary for maintenance of the population in neurons (Aim 1). Independently, we designed a strategy to define the mechanism of motor-mitochondria attachment specifically necessary for retrograde transport of the organelle (Aim 2). Together, the proposed experiments will provide mechanistic insight into how and why mitochondria move in the retrograde direction while also defining the source of healthy organelles necessary for maintenance of the mitochondrial population in neurons. The knowledge gained will enhance our insight into the basic biology of the cell that can be repurposed for potential therapeutic interventions.

NIH Research Projects · FY 2026 · 2022-02

ABSTRACT Alzheimer's disease (AD) is the 6th leading cause of death in the United States and its prevalence continues to rise. AD has no clinically available curative treatments and findings from active clinical trials testing novel disease-modifying therapeutics have thus far been disappointing. There is, therefore, a growing urgency to identify early markers of AD, causative factors leading to dementia, and alternative treatment approaches for halting the global crisis posed by this debilitating condition. Cardiovascular disease, as well as cerebrovascular disease (CVD), has a strong link with both mild cognitive impairment and AD dementia; however, the question of whether CVD modulates underlying pathophysiology of AD has only recently begun receiving attention. To provide insights into AD relationships, non-invasive Magnetic Resonance Imaging (MRI) is being utilized in longitudinal studies of AD risk-enriched populations. The present project goes far beyond currently available MRI techniques which lack sensitivity and specificity to address key vascular hypotheses in AD. MRI methods commonly employed today such as fluid attenuation and susceptibility imaging only indirectly measure CVD and cannot inform on the dynamic vascular motion and hemodynamic phenomena that have been indicated in animal models to affect AD pathology. To address these gaps, the overarching objective of this project is to enable characterization of cerebrovascular involvement in AD through the development and study integration of a novel battery of non-invasive, MRI-based measures of cerebrovascular health. Building upon foundational studies at our institution, this work proposes innovative MRI technology to improve characterization of CVD in AD, specifically vascular stiffening and its relationship with cerebrovascular flow dynamics. We propose an ensemble of motion encoded MRI techniques which provide detailed depiction of autoregulatory flow dynamics and vascular stiffness in both the macro and micro vasculature. In this project, the novel methods will be technically developed harnessing deep learning from vast prior imaging data, validated with optical imaging, and characterized in healthy human subjects. We then will obtain key data characterizing cerebrovascular changes in a study of AD biomarker confirmed subjects with the overall goal of identifying the modifying effect of vascular disease on the symptom expression of cognitive impairment, AD biomarker accumulation, and neurodegeneration. Our pilot data suggest subjects with AD have a premature increase in arterial stiffness and decreased fluctuations in cerebral blood flow. Upon completion, this study will provide insights into which specific aspects of CVD are primary factors moderating AD interactions. Participants targeted for this study have extensive existing AD biomarker data and are being followed longitudinally through studies within the Wisconsin Alzheimer's Disease Research Center. The methodologies will be uniquely positioned for incorporation into large longitudinal cohort studies investigating AD mechanisms and evaluating putative risk and protective factors.

NIH Research Projects · FY 2026 · 2022-02

ABSTRACT Influenza virus is a serious public health threat causing high levels of morbidity and mortality. The annual disease burden from influenza places a significant strain on our healthcare infrastructure and economy. This is despite many efforts to control disease with yearly vaccination, antiviral drug therapies, and other medical and public health interventions. To address the continuing health and economic costs, there is a clear need to better understand the molecular mechanisms of influenza virus replication and how these can be manipulated for therapeutic benefit. Influenza virus exploits, and in some cases subverts, cellular factors and pathways to promote replication. We identified RNA:protein interactions between viral and host partners that impact innate immune responses during infection. We showed IFIT2 is a critical cellular protein that binds viral mRNAs to enhance replication. This was surprising, as IFIT2 is one of the first proteins expressed in response to viral infection and displays broad-spectrum antiviral activity. The mechanisms underlying the antiviral activity of IFIT2, and how this is co-opted into a proviral effector by influenza virus, are not yet known. We also showed that influenza nucleoprotein (NP) is a key viral protein that binds host RNAs. This assigns a new activity to NP that our data suggest is part of a previously unappreciated strategy to dampen innate immune responses. The overall goal of this application is to determine how these RNA:protein complexes composed of both viral and host components manipulate innate immune responses to support viral replication. In Aim 1, we investigate how IFIT2 functions as a front-line defender in a broadly acting antiviral response. We hypothesize that IFIT2 enhances translation of antiviral proteins, an event that is repurposed by influenza virus to promote production of viral proteins. We test this using experiments that investigate the processes by which IFIT2 engages target RNAs, affects translation of its bound mRNAs, and alters infection. Aim 2 interrogates the impact of interactions between viral NP and host RNAs. This aim proposes that NP:RNA complexes moderate innate immune responses. We investigate this by studying RNAs and infection-induced events that activate innate immune pathways, and discern the impact on viral replication. The results from this proposal will establish a mechanistic understanding of the viral and host factors regulating innate immune responses and how influenza virus tips the balance to favor replication. Moreover, while our studies focus on influenza virus, the underlying mechanisms we discover will have broad impacts on the general understanding of host antiviral responses and unexpected strategies used by viruses to counteract them. Completion of this proposal will provide fundamental knowledge that can contribute to new therapeutic approaches or cellular targets that can be exploited for the rational development of anti-influenza virus therapies.

NIH Research Projects · FY 2026 · 2022-02

Project Summary/Abstract Dental settings have been plagued with persistent no-show rates that are associated with low quality of care, inadequate care access, and inefficient use of organizational resources for the patients and organizations that need them most. Patient appointment no-shows are particularly pronounced in organizations that service underserved populations. Studies documenting strategies to reduce no-shows in dental settings are limited, with organizations reporting difficulty in replicating the findings, particularly in diverse patient populations. In this study, a team of researchers from the Marquette University School of Dentistry and the Center for Health Enhancement Systems Studies (the Center) at the University of Wisconsin–Madison College of Engineering will test the effectiveness of three no-show reduction strategies using an innovative full factorial design that allows for the simultaneous testing of different combinations of the no-show strategies. The intervention strategies include a) using Motivational Interviewing techniques when scheduling appointments, b) conducting reminder calls 24 and 48 hours before an appointment, and c) applying open-access scheduling, along with other practices found to reduce no-shows in other sectors of health. The practices to test emerged from a pilot conducted by the study team with dental clinics that primarily serve underserved populations. A significant aspect of this study is the use of the NIATx Organizational Change Model (the NIATx model) to facilitate and provide a standardized evidence-based strategy for no-show practice implementation and to address the concern of the inability to apply proven no-show strategies. The UG3 will require a full two years to finalize the study protocol and operations manual and formalize partnerships with 40 dental clinics. The UH3 will test if different combinations of the no-show strategies of Motivational Interviewing, reminder calls, and scheduling practices can improve no-show rates compared to treatment as usual (Aim 1). A mediational analysis will examine whether using the NIATx model with fidelity and Organizational Readiness for Change mediates no- show rates (Aim 2). The qualitative portion of the study will aid in interpretation of quantitative results and will be used to gain a more in-depth understanding of the factors that promote or impede the implementation of the no-show strategies (Aim 3). This trial will be among the first RCTs to test a combination of no-show strategies in health care that is focused on the underserved populations where no-shows are most pronounced and have the greatest adverse clinical consequences. The investigative team has successfully conducted a pilot study on this subject and has considerable experience with large clinical trials. In summary, the study intends to provide an evidence-based replicable approach to reduce no-show rates, with the goal of helping the dental care field make organizational changes that support evidence-based care and improve care access.

NIH Research Projects · FY 2026 · 2022-02

While scientific and medical advances have dramatically increased our ability to prevent, detect and treat cancer, gaps remain regarding the impact of environmental exposures—particularly chemical and physical factors—on cancer risk. The overarching goal of this application is to build collaborative infrastructure and facilitate transdisciplinary scientific research for enhancing our understanding of environmental exposures influencing cancer etiology, and the genetic, behavioral, and structural factors that modify risk across diverse populations. We propose to serve as the Coordinating Center for the Cohorts for Environmental Exposures and Cancer Risk (CEECR) Program. Our mission is to provide intellectual leadership and logistical and collaborative infrastructure for the CEECR program by integrating efforts across the CEECR cohorts, facilitating research activities, identifying opportunities for cross-CEECR collaboration, and disseminating research findings through partnerships with stakeholders. We will draw upon extensive experience coordinating large-scale consortia, conducting prospective cancer epidemiology studies, and engaging with community partners to support the CEECR Program. We will facilitate cooperation across the CEECR cohorts to achieve their UG3/UH3 milestones and realize their full potential as an integrated sustained powerful source of knowledge for characterizing the impact of environmental exposures on cancer risk. Specific Aims include: 1) providing intellectual leadership and multidisciplinary scientific expertise, establishing a governance structure, and identifying opportunities to catalyze cross-cohort collaboration; 2) assisting with the identification of common data elements and the integration of research efforts regarding biological samples and innovative technologies; 3) facilitating innovative transdisciplinary research by developing evidence-based infrastructure to support collaboration, coordination, communication, outreach and engagement, and career enhancement; and 4) fostering resource sharing by CEECR members with external researchers and stakeholders—including the deposition of data in NIH repositories—to accelerate research and move findings towards translation and prevention. Our multidisciplinary team includes experts in cancer epidemiology, environmental health, team science, cancer health disparities, cancer biology, toxicology, data science, applied public health practice, outreach/community engagement, and research administration. The University of Wisconsin Carbone Cancer Center provides the ideal environment for supporting the Coordinating Center due to its resources and established relationships with local and national research and advocacy organizations. The CEECR Coordinating Center will use multiple innovative approaches to leverage program members’ common passion for elucidating the role of environmental factors in cancer etiology, provide expanded opportunities for collaboration, and enable the CEECR Program to bring about substantive reductions in the cancer burden paying special attention to populations historically excluded from the full benefits of scientific advances.

NIH Research Projects · FY 2026 · 2022-01

ABSTRACT Down syndrome (DS) is a leading known cause of intellectual disability and a highly recognized genetic syndrome that involves multiple medical co-morbidities. Hearing deficits in DS are estimated to occur up to rates of 80-90% and are thought to be caused by a combination of structural and functional abnormalities in the external, middle and/or inner ear. This project aims to tackle a timely and significant question regarding the role of hearing loss in DS on auditory function, cognition, language, and structural integrity of brain regions that are important for hearing. Despite the known pervasive nature of hearing deficits in DS, to date, research has yet to identify specific consequences of hearing deficits associated with trisomy 21. Aim 1 will assess hearing status, auditory integrity and maturation using objective measures of auditory processing in subcortical auditory brainstem responses (ABR) and cortical auditory evoked potentials (CAEPs), and functional processing behavioral measures of speech identification and sound localization. The goal is to understand how hearing loss impacts auditory processing and auditory maturation in DS is after compensating for hearing loss. Aim 2 will investigate the associations between hearing/auditory deficits and overall intellectual functioning as well as memory, attention, and executive function, as these cognitive domains have been shown to be influenced in critical ways by hearing status and auditory impairments in non-DS populations. Aim 2 will also examine the associations between hearing and auditory function and receptive and expressive vocabulary, obtained via standardized measures and language samples. Aim 3 will investigate the associations between hearing/auditory deficits and imaging measures of structure and microstructure in brain regions involved in auditory processes, focusing on regional morphometry, relaxometry, and microstructural diffusion measures. We aim to understand whether abnormalities are domain specific within auditory processes, global to all auditory brain regions, or generalized/global to DS brains but not auditory-specific, i.e., related to issues from generic pathophysiology in DS. This work meets the programmatic objectives of INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndrome (INCLUDE) and will advance understanding of the consequences of hearing loss in DS for functional auditory, cognitive and language outcomes and brain integrity. One immediate benefit is that results will inform clinical assessment and intervention of hearing loss catered specifically to the pathophysiology in DS. Longer-term impact is the identification of measures to evaluate effectiveness of future intervention of hearing loss in DS.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY The overall goal of this multi-institutional, multi-disciplinary grant is to bring histotripsy closer to widespread human clinical use by developing a C-arm based platform to plan, target, and assess histotripsy ablations. Histotripsy is a unique noninvasive, nonthermal, nonionizing and highly precise ablation modality that has recently undergone a successful phase I clinical trial. Rodent studies demonstrate the promise of histotripsy to potentiate an anti-cancer immune effect and combine synergistically with check-point inhibition. Although histotripsy has highly promising therapeutic capability, a major barrier to adoption is the lack of a reliable method for visualizing and targeting tumors. Systems to date have only used conventional ultrasound imaging for targeting the tumor and monitoring and assessing the ablation zone. Unfortunately, ultrasound is operator dependent, inherently limited for evaluating 3D tumors and ablation zones, unable to penetrate certain biologic tissues such as bone and gas, and is of limited use in patients with large body habitus. Conventional CT and MRI are not ideal for histotripsy due to limited space and field of view as well as artifacts from the therapeutic transducer and robotic arm. C-arm x-ray fluoroscopy with cone-beam CT (CBCT) is an excellent option for targeting during histotripsy and a C-arm based platform would complement current US guidance techniques. C-arms are ubiquitous with world-wide distribution and expertise, have an open design with easy access to the patient, provide volumetric data from CBCT for treatment planning and ablation zone assessment, and 2D fluoroscopy can be adapted to deliver real-time automatic and accurate targeting. To advance histotripsy toward widespread human clinical use, we will develop a C-arm fluoroscopic and CBCT based approach that can be used to plan, target, and assess histotripsy ablations. Aim 1 of this grant will be to develop and validate C-arm based algorithms to accurately and automatically target a specific volume of tissue for histotripsy ablation. To improve the precision of histotripsy treatments, we will then develop respiratory motion compensation techniques in Aim 2. Aim 3 will develop a deep-learning approach to compensate for the variable speed of sound through different biologic tissues (analogous to aberration correction). In Aim 4, we will integrate the developments from Aims 1-3 and perform prospective validation studies in human-scale porcine and rabbit VX2 tumor models to determine the accuracy, efficacy and safety of C-arm guided histotripsy as well as its effect on survival. If successful, this grant will catalyze the use of C-arm fluoroscopy with CBCT to plan, target, and assess histotripsy of liver tumors, and bring the technique closer to widespread human clinical use.

NIH Research Projects · FY 2025 · 2022-01

Project Summary/Abstract We aim to improve the cure rates for metastatic cancers. To achieve this we propose a combined modality approach to stimulate and diversify an endogenous anti-tumor immune response at all tumor sites to recognize and destroy tumor cells in a manner that will prevent recurrence and enable long-term cancer free survival. Immune checkpoint inhibitors (ICI; e.g. anti-PD-L1), are a class of immunotherapies that modulate immune tolerance of a tumor by blocking specific inhibitory receptor-ligand interactions on the surface of T cells and thereby overcoming T cell inhibition or exhaustion. In patients with immunologically “hot” tumors, characterized by a pre-existing but exhausted anti-tumor immune response, ICIs can restore efficacy to the anti-tumor immune response, sometimes resulting in complete and durable tumor regression. However, ICIs have not shown clinical benefit in the treatment of immunologically “cold” cancers that are characterized by low levels of T cell infiltrate and low mutation burden resulting in few mutation-created neo-antigens. To overcome immunotherapy treatment barriers posed by immunologically cold tumors, we propose to combine systemic delivery of ICIs with systemic delivery of radiation by targeted radionuclide therapy (TRT). To date, nearly all approaches to combining radiation and immunotherapy have used external beam radiotherapy (EBRT), which promotes tumor immune cell infiltration through activation of type I interferon (IFN) responses. Administration of EBRT to multiple tumor sites or to the whole body (to target radiographically occult or microscopic disease) would result in prohibitive toxicity, including lymphopenia. TRT is a systemic method of delivering a therapeutic radionuclide to a tumor, which poses an alternative option for delivery of immunomodulatory radiation to metastatic tumor sites without causing immunosuppression. The Weichert lab at the University of Wisconsin-Madison has developed a novel class of TRT, known as NM600, an alkylphosphocholine analog that is selectively taken up and retained in nearly any tumor type in any location. Our broad hypothesis is that unique physical properties of radionuclides (e.g. emission type, linear energy transfer, half-life, tissue range) differentially impact immunomodulation by TRT. In this study, the immunomodulatory capacity of alpha- (225Ac) and beta- (90Y) particle emitting radionuclides will be compared directly. In a project that builds upon the ongoing collaborative progress of the Morris and Weichert labs, we will now determine the radionuclide-specific potency of combining TRT with immunotherapy to enhance the immune response against immunologically cold tumors. In murine models, we will: 1) expand on preliminary data showing potent synergy with the combination of TRT and ICI, 2) evaluate therapeutic mechanisms of TRT and ICI using the intrinsic properties of 225Ac- and 90Y-NM600, focusing on type I IFN response activation and 3) investigate potential enhanced tumor responses with the combination of two distinct radionuclides with ICI. The insights and treatment regimens developed in these studies should enable rapid translation to clinical testing in patients and potentially for any type of metastatic cancer.

NIH Research Projects · FY 2026 · 2022-01

Project Summary/Abstract Vocal fold (VF) hyperkeratosis – an accumulation of epithelial surface keratin resulting in clinical leukoplakia – is primarily managed using destructive techniques that risk iatrogenic injury, fibrosis, and voice impairment. Given the potential morbidity of biopsy and lesion removal, there is a compelling need for new approaches to the workup and treatment of VF leukoplakic disorders that are non-destructive and grounded in disease pathophysiology. In the current application, we propose translational research that will advance this goal. Our preliminary data indicate that the essential nutrient vitamin A is a key regulator of epithelial health and that systemic vitamin A deficiency can directly contribute to hyperkeratosis. Based on evidence of vitamin A’s importance to VF stellate and epithelial cell biology, and its direct relevance to VF hyperkeratosis, we propose three Specific Aims that will advance this research area towards greater clinical applicability by: (i) assessing the systemic vitamin A status and lesion histopathology of patients with VF hyperkeratosis, and (ii) examining vitamin A trafficking to, and uptake by, normal and hyperkeratotic VF mucosa in a rat model. Our overarching goal is to determine if there is a pathophysiologic rationale for vitamin A supplementation in the treatment of VF hyperkeratosis. Such a therapy would be transformative – both as a first-line approach for patients who require conservative management, as well as an adjuvant therapy in patients for whom surgical treatment is indicated.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY / ABSTRACT Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a complex inherited disorder of the heart produced by mutation in proteins of the desmosome, such as plakophilin-2 (PKP2). Cardiac arrhythmias, and potentially sudden death, often occur in ARVC patients during the early stages of the disease, a “concealed phase” that presents before the onset of structural cardiomyopathy. The molecular and cellular mechanisms of these arrhythmic events remain unclear, hindering the search for effective strategies to treat patients. My long-term goal is to delineate the mechanisms of arrhythmia in ARVC and to identify potential drug targets to prevent sudden cardiac death. Mice with tamoxifen (TAM)-induced ablation of PKP2 (PKP2cKO) develop a phenotype evocative of human ARVC: a concealed stage with high incidence of arrhythmia but without structural remodeling at 14 days, cardiomyopathy of right ventricle dominance at 21 days, and biventricular cardiomyopathy, heart failure and death at ~42 days post-TAM. We reported that PKP2cKO hearts show significant dysregulation of Ca2+ handling at different stages of disease progression but, most remarkably, during the concealed stage of the disease. This proposal aims to elucidate the mechanisms underlying cardiac arrhythmia in PKP2-deficient hearts focusing on the microdomain where Ca2+ regulation takes place. I hypothesize that dysfunction of the cardiac ryanodine receptor (RyR2), a major intracellular Ca2+ release channel, and the ensuing Ca2+ mishandling are critical triggers of cardiac arrhythmia in the PKP2cKO mouse and, hence, in ARVC. These aims will test my hypothesis: 1) Determine the role of protein kinase C (PKC) phosphorylation in the regulation of RyR2 channel function and calcium homeostasis. Preliminary data suggest that RyR2 is undergoing phosphorylation in PKP2cKO hearts at Thr2810. This previously uncharacterized site is a predicted PKC substrate. I hypothesize that PKC phosphorylation of RyR2 at Thr2810 regulates channel function and contributes to arrhythmogenic Ca2+ release in the diseased heart. 2) Define the contribution of RyR2 dysfunction in the onset and progression of heart disease in PKP2cKO mice. Preliminary data suggest that RyR2 phosphorylation at Thr2810 and Ser2030 is increased in ARVC. I hypothesize that inhibition of RyR2 phosphorylation at these sites prevents arrhythmia and sudden death in PKP2cKO mice. 3) Test the efficacy of RyR2 modulators for the prevention of arrhythmia in PKP2-deficient hearts. I hypothesize that pharmacological modulation of RyR2 is beneficial to prevent arrhythmia in PKP2cKO mice and hearts. The completion of these aims will provide significant insight into the regulation of RyR2 function in a model of PKP2cKO deficiency and hence shed light on the mechanisms underlying ARVC. I anticipate these results will advance the status of RyR2 as a potential therapeutic target to reduce the risk of arrhythmias and increase life-expectancy of patients with ARVC.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT Synthetic photochemistry is a powerful tool for biomedical research. The small-molecule therapeutics that constitute the core of modern molecular medicine occupy a relatively narrow segment of chemical diversity space. Photochemistry offers a new capability that can expand the range of chemical structures that can be investigated for their biological activity and their potential as new life-saving drugs. Specifically, reactions that are driven by light can use the energy of a photon to produce highly reactive intermediates that react in ways that are not accessible by other means. Our group's research focuses on the discovery of reaction methods that can control the outcomes of photochemical processes in predictable ways. We have a long-standing interest in the development of mechanistically novel catalytic processes that are directly applicable to the synthesis of complex bioactive compounds. We are also interested in the use of external stimuli that can control or divert the intrinsic reactivity of open-shelled photogenerated intermediates towards desirable synthetic goals. Finally, we are deeply committed to elucidating the mechanisms by which photochemical transformations occur. The next phase of our research will continue to investigate these broad themes. We propose investigations into new strategies for controlling the stereochemistry of diverse photochemical reactions. We will study a new approach towards the generation of carbocationic intermediates from photoredox activation. Finally, we will develop a new class of photoreactions that take advantage of the direct photochemistry of base metal coordination complexes. These studies will result in new, generalizable strategies for the controlled photochemical synthesis of complex organic molecules.

NIH Research Projects · FY 2026 · 2021-12

ABSTRACT Diffuse large B cell lymphoma (DLBCL), which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive and less curable. More than 50% of patients with ABC DLBCL are refractory to or relapse from current frontline immunochemotherapy. Clinical use of ibrutinib, a selective inhibitor for Bruton tyrosine kinase (BTK) in the B cell receptor (BCR) signaling pathway, has achieved an initial response rate of 30%-40% in refractory/relapsed ABC DLBCL. Primary and acquired drug resistance, however, are still significant and impact the long-term survival of more than 60% of these patients. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. The BCR and JAK1/STAT3 signaling pathways are essential for the survival and proliferation of ABC DLBCL cells. We discovered that EGR1 is a converged downstream target of both pathways in ABC DLBCL and the level of EGR1 expression is elevated in ABC DLBCL compared with normal human tonsils and lymph nodes. We revealed novel mechanisms of EGR1 in transcriptional activation and repression of target genes with strong translational impact in treating aggressive lymphoma. EGR1 mediates transcriptional activation through the p300/H3K27ac/BRD4 axis to induce MYC expression and activate MYC target genes. Synergistic inhibition of cell growth was observed between EGR1 shRNA and AZD5153, a novel BRD4 inhibitor that is currently under clinical investigation. On the other hand, EGR1 mediates transcriptional repression of the type I interferon pathway genes, expression of which otherwise causes cancer cell death. Consistently, EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. Using newly derived, ibrutinib-resistant ABC DLBCL cell lines, we demonstrated that EGR1 is among the most highly expressed genes relative to ibrutinib-sensitive parental cells, and co-targeting of BRD4 and interferon signaling inhibits growth of ibrutinib-resistant cells in vitro and in vivo. Based on these discoveries, the central hypothesis is that EGR1 is a unique oncogenic driver orchestrating multiple important signaling pathways and represents therapeutic vulnerability in patients with ABC DLBCL, especially for those with ibrutinib resistance. To test our hypothesis, we will pursue the following specific aims: (1) Elucidate effects of EGR1 on oncogenesis; (2) Establish the role of EGR1 in ibrutinib resistance; and (3) Co-target EGR1 downstream BRD4 and type I interferon signaling to overcome drug resistance in DLBCL. Dissecting the transcriptional activation and repression modules of EGR1 in DLBCL tumorigenesis and ibrutinib resistance is essential because the novel mechanistic insights will provide a molecular basis for developing the most effective therapeutic strategy for treatment of DLBCL patients, including those with ibrutinib resistance.

NIH Research Projects · FY 2026 · 2021-12

Project summary. During lytic infection, the AIDS-associated tumor virus Kaposi’s sarcoma-associated herpesvirus (KSHV) blocks cells from activating the anti-viral type I interferon (IFN) responses. This block of the innate immune response facilitates efficient viral replication, which in turn contributes to development of Kaposi’s sarcoma. Thus, elucidating the mechanisms by which KSHV evades the host innate immune response may provide insights on how to target this and other KSHV-induced tumors. However, because of the complex and redundant nature of the type I IFN induction pathway, how KSHV blocks this early antiviral response is still incompletely understood. In a previous study, we found that the host protease caspase-8 is a major mediator of type I IFN inhibition by KSHV. KSHV reactivation from latency only triggers minimal type I IFN induction, but there is a much stronger transcriptional induction and secretion of type I IFNs when caspase-8 is also inhibited. This stronger IFN induction, in turn, reduces KSHV reactivation. These results indicate that caspase-8 activity is necessary to inhibit IFN induction, and thus promotes KSHV replication. This finding was surprising because caspase-8 activation is generally considered antiviral as it induces apoptotic cell death. However, we do not detect wide-spread cell death during reactivation from latency despite caspase-8 activation, suggesting that caspase-8 is hijacked and repurposed by KSHV to inhibit type I IFN responses. At present, the molecular mechanisms that lead to caspase-8 activity and the pathways that are targeted by caspase-8 to control type I IFN during KSHV infection remain unclear. We have new preliminary data suggesting that caspase-8 is activated by a pathogen sensing pathway, the Toll-like receptor (TLR) pathway, as a cellular response to infection. Caspase-8 then proceeds to inhibit a different pathogen sensing pathway, cGAS-mediated DNA sensing. Therefore, we hypothesize that KSHV is taking advantage of a TLR-mediated cellular response to infection that activates caspase-8. KSHV is then able to redirect this activity to inhibit DNA sensing instead of activating apoptosis. We will test this hypothesis and determine how caspase-8 is activated by TLR signaling in KSHV-infected cells without triggering cell death (Aim 1), and which host protein(s) are cleaved by caspase-8 to block cGAS-induced type I IFN responses (Aim 2). Moreover, we will also investigate whether and how caspase activity is connected to other previously described mechanisms of immune evasion by KSHV (Aim 3). As caspase-8 is a druggable target, understanding how caspase-8 is used by KSHV to regulate type I IFNs and promote its replication will reveal whether and how this enzyme could be exploited for KSHV therapy. This is important as there are no target therapies for this virus, and Kaposi’s sarcoma remains one of the leading types of cancers in sub-Saharan Africa and the second most common AIDS-associated malignancy in the US. This project will also uncover fundamental aspects of caspase signaling that may play a role in other diseases connected to IFN.

NIH Research Projects · FY 2025 · 2021-12

Project Summary/Abstract Functionally mature beta cells are essential to glucose homeostasis and their loss or dysfunction underlies all types of diabetes mellitus. In recent years, it has become clear that not all beta cells are permanently lost in either type of diabetes. Instead, chronically stressed beta cells lose their functionally mature phenotype and shift to a dysfunctional state in a process called de-differentiation. Preventing or reversing beta cell de- differentiation represents a promising approach to restoring functionally mature beta cell mass in diabetics. We have recently identified five novel genetic regulatory networks that we hypothesize to be important for regulat- ing mature beta cell function and identity. The overarching goal of this proposal is to establish the above five regulatory networks as novel genetic and pharmacological switches for controlling beta cell function and identi- ty. In Aim 1, we will map in detail each of the networks by determining direct and indirect regulated nodes for each regulator. We will further identify the networks' cellular function and find upstream signaling pathways predicted to affect the networks' behavior in both human and mouse beta cells, and their points of perturbation during beta cell de-differentiation. In Aim 2, we will test the causality of the five predicted regulators on beta cell function and identity in vitro in primary mouse and human islets, and in vivo, using genetic mouse models where available. We will further test the effect of genetic intervention points in the networks to lock in place ma- ture beta cell identity under the different diabetogenic stresses.

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically heterogeneous sample of the US population. To accomplish this, the Healthy Brain and Child Development (HBCD) Consortium has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,200 mothers and infants enrolled at 27 sites across the United States (US). The HBCD Study will carry out a common research protocol under direction of the HBCD Consortium Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD Data Coordinating Center (HDCC). The overarching goal of the HBCD Study is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a varied cohort that is representative of the US population; 2) pregnant woman with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD Study will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state-of-the-art methods. The HBCD Study will inform public policy to improve the health and development of children across the nation. This study is part of the NIH’s Helping to End Addiction Long-term (HEAL) initiative to speed scientific solutions to the national opioid public health crisis. The NIH HEAL Initiative bolsters research across NIH to improve treatment for opioid misuse and addiction.

NIH Research Projects · FY 2025 · 2021-09

Summary Angiogenesis is required for proper development of the embryonic circulatory system and is an important step in the progression of many eye diseases, including retinopathy of prematurity (ROP). Therefore, understanding how the normal regulatory mechanisms in the retina keep angiogenesis in check has great clinical implications. We recently showed global deletion of Bim, a proapoptotic member of Bcl-2 family of proteins, protects the developing retinal vasculature from hyperoxia–mediated vessel obliteration and ischemia-mediated neovascularization in the preclinical mouse model of ROP, the oxygen-induced ischemia retinopathy (OIR) model. In addition, we showed that targeted deletion of Bim in retinal endothelial cells, pericytes or astrocytes does not protect the developing retinal vasculature from exposure to hyperoxia–mediated vessel obliteration or from ischemia-mediated neovascularization. These results strongly support an important role for Bim expression in retinal inner neurons in proper regulation of developing retinal vasculature. Our hypothesis is that Bim expression in retinal neurons plays a central role in deriving the sensitivity of developing retinal vasculature to hyperoxia–mediated vessel obliteration and ischemia–mediated retinal neovascularization during ROP. In Aim 1, we will determine the contribution of Bim expression in the inner retinal neurons to hyperoxia-induced vascular damage. In Aim 2, we will determine the contribution of VEGF expression in the inner retinal neurons to hyperoxia-induced vascular damage. Understanding how Bim expression in retinal neurons regulates retinal vascular development and enhanced sensitivity to hyperoxia will provide insight into Bim mechanisms of action and aid in the development of alternative ways to modulate retinal angiogenesis.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract The project addresses vascular contributions to cognitive impairment and dementia (VCID) using a design that exploits the enrollment of persons with the autosomal dominant gene for Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Exploration of VCID by focusing on the rare heritable monogenic disorder, CADASIL, will advance knowledge of the full spectrum of vascular dementia from presymptomatic gene carriers through dementia and will facilitate an examination of mixed dementia in CADASIL participants also having AD. This research is novel from any other in its efforts to study a single-gene vascular dementia group throughout the life span in an effort to reduce vascular dementia heterogeneities selecting persons enriched for certain future vascular disease secondary to NOTCH3 gene mutation. CADASIL is caused by a mutation in the gene NOTCH3, which encodes a receptor protein. Although rare, CADASIL is the most common heritable cause (due to a single gene mutation) of vascular dementia. Many consider CADASIL to be a good single gene model of small vessel disease and vascular dementia. By improving our understanding of CADASIL and its progression, we will be better able to understand VCID as well as mixed dementias having both CADASIL and the most common sporadic dementia, Alzheimer's disease. This is particularly important because of the fact that many cases of Alzheimer's disease have co- occurring vascular dementia. More than 200 genetic variations in NOTCH3 have been identified, but it is not known which cause milder, or more severe, forms of VCID. Furthermore, among the NOTCH3 mutations known to cause CADASIL, and even within CADASIL families, there is wide variability in age at onset, disease progression, brain imaging abnormalities and presentation of symptoms. Some recent data reported by the CADASIL European Consortium suggests that patients with mutations in certain parts of NOTCH3 cause a more severe form of CADASIL than do mutations in other parts of the gene. It's also likely that variations in other genes influence the effect of NOTCH3 mutations on VCID. Unfortunately, no large cohort of CADASIL patients for clinical research has been organized in North America. Better understanding of the associations between clinical presentation, potential disease modifiers (i.e., risk factors) and NOTCH3, as well as other gene variations in a new cohort in the United States could improve understanding of the causes and severity contributions to various forms of small-vessel disease and vascular dementia. To accomplish this, we will recruit, characterize and follow longitudinally a cohort of 400 NOTCH3 variant/mutation carriers, plus 100 non- carriers as controls, from U.S. CADASIL families. Participants will be evaluated every 18 months with detailed neurological, cognitive, behavioral, functional, blood, genetic and brain MRI assessments. This study will provide critical information regarding CADASIL that will also advance understanding for the most common type of mixed dementias (viz., VCID and AD).

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Among working-age U.S. adults, diabetic retinopathy remains the leading cause of blindness despite landmark National Eye Institute (NEI) clinical trials showing that early detection and treatment reduce the risk of severe vision loss by over 90%. Yet adherence with yearly diabetic retinopathy screening in the U.S remains below half of patients in rural communities. Ocular telemedicine (i.e. teleophthalmology) can substantially increase diabetic retinopathy screening rates and prevent blindness. However, there is often very limited use of this technology even after a teleophthalmology program is established in multi-payer health systems, where the majority of Americans receive their care. To overcome major barriers identified for teleophthalmology use in rural multi-payer primary care clinics, the PI developed and piloted Implementation to Sustain Impact in Teleophthalmology (I-SITE) in a NEI K23 Career Development Award. I-SITE is the first implementation program specifically designed to overcome barriers to teleophthalmology use by tailoring its integration into rural primary care clinic workflows. The proposed study is a multi-center randomized controlled trial aiming to: (1) test the effectiveness of I-SITE, (2) identify explanatory factors and implementation components that distinguish high and low teleophthalmology use in rural health systems following I-SITE implementation, and (3) evaluate implementation costs. We hypothesize that I-SITE will sustain significant diabetic eye screening rate increases at 18 months compared to usual care teleophthalmology. This UG1 proposal directly responds to multiple elements of the NEI Strategic Plan, including areas of emphasis in telemedicine, diabetic retinopathy, and expanding access to eye care in rural populations. This research will facilitate the effective translation of telemedicine technology in rural multi-payer primary care clinics to improve diabetic retinopathy screening rates. The effective integration and scale-up of ocular telemedicine would greatly expand eye care access for millions of rural Americans. This will be critical for preventing avoidable blindness by overcoming the shortage of eye care providers and meeting the increased demand for eye screening resulting from major projected increases in diabetes prevalence. As we face unprecedented challenges to our nation's health systems, never before in our history has the need for a rapid transition to telehealth been more acute. Our study will provide vital knowledge regarding the methods and factors needed to successfully translate telehealth technology into widespread clinical practice for improving public health nationwide.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Falls are the leading traumatic cause of both injury and death among older adults. American emergency departments (EDs) see over 3 million fall victims yearly, yet they play little role in primary or secondary fall prevention. The ED is an ideal site to identify patients at risk of future falls, however in this setting preventive care cannot be implemented at the expense of the primary mission of the ED: the provision of emergency care in a time-pressured environment. As the population ages, and the ED continues to expand its role as the primary site for delivery of acute unscheduled care, there is an urgent need to create a scalable intervention to assess older adults for fall risk and link them to appropriate risk reduction interventions after discharge without adding additional workload for nurses or physicians. Through an AHRQ K08, our study team has developed and validated an innovative automated screening and referral intervention for fall risk. This intervention harnesses existing data to select and connect patients to appropriate primary and secondary prevention services after ED visits without adding burden to nurse or physician workloads. This intervention features smart use of automation for screening and referral tasks maintaining physician decision autonomy, as well as the unique ability to adjust referral rates based on clinic availability. This intervention features smart use of automation for screening and referral tasks maintaining physician decision autonomy, as well as the unique ability to adjust referral rates based on clinic availability. Based on our work, UW Health is currently piloting the intervention, and has committed to implementing it at three diverse ED sites. This study will adapt the intervention for implementation at additional sites, and investigates the implementation and effectiveness of the automated screening and referral process in all three EDs through three specific aims: 1) Adapt the design of an automated screening and referral intervention for implementation in three diverse ED settings, using a human factors approach. 2) Test the effectiveness of the automated screening and referral intervention on both completed referrals to a multidisciplinary fall prevention clinic and rates of injurious falls using EHR data generated during implementation. 3) Evaluate implementation of the automated screening and referral intervention in three diverse ED sites using a mixed methods approach. This grant proposal builds upon our previous innovative work developing both CDS and risk- stratification algorithms to improve the quality and safety of care delivered to older adult ED patients. We will address the urgent and growing need for a scalable strategy for fall risk reduction from the ED by demonstrating the effectiveness of our novel approach in a study spanning diverse hospital types and patient populations. Furthermore, knowledge gained from this work will inform other use cases which could benefit from automated risk-stratification and care coordination in the ED and beyond.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in over 26 million infections and overwhelmed healthcare systems throughout the U.S. The novel nature of COVID-19 has generated unprecedented diagnostic and therapeutic dilemmas. One area of emerging concern is the collateral impact of the pandemic on increased antibiotic prescribing and an associated acceleration of bacterial resistance. For instance, early reports indicate that a high percentage of patients hospitalized with COVID-19 receive antibiotics despite few having confirmed bacterial co-infections. In addition to the public health implications, overuse of antibiotics is also a threat to patient safety due to the risk of serious adverse drug events and Clostridioides difficile colitis. In January 2021, the Society for Healthcare Epidemiology of America issued a white paper outlining research priorities related to COVID-19 that highlighted an urgent need to “identify the impact of changes in health care utilization and delivery on antibiotic prescribing” and “develop and implement optimal Antimicrobial Stewardship Program (ASP) strategies to improve antimicrobial use and patient outcomes while adapting to changing healthcare delivery during COVID-19”. This project is specifically designed to address this call to action as we aim to comprehensively characterize the impact of the COVID-19 pandemic on antibiotic prescribing and bacterial resistance trends in acute care hospitals and identify strategies that effectively promote resilient antibiotic stewardship. The assembled team is uniquely qualified to conduct this project given our expertise in evaluating antibiotic prescribing patterns, access to data from ~350 U.S. hospitals and extensive experience using systems engineering methods to analyze stewardship interventions. For the quantitative analyses, we will first characterize overall and condition specific antibiotic prescribing trends before and after COVID-19 using an interrupted time series analysis. Next, we will identify patient and hospital level factors that increased the risk of non-indicated antibiotic prescribing during the COVID-19 pandemic, with the goal of identifying potential intervention targets. Finally, we will complete a systems engineering guided qualitative analysis, focused on hospitals that least and most effectively mitigated the impact of COVID-19 on antibiotic prescribing, to identify systems-level contextual factors and strategies. These results will be used in a multidisciplinary co-design process to develop an antibiotic stewardship implementation toolkit that enhances resiliency during operational upheaval and is transferable between organizations. Given the dynamic nature of the pandemic (e.g. variant strains), it is imperative to classify the previous, ongoing and future adverse impacts on antibiotic prescribing to guide development of tailored stewardship strategies for widespread dissemination. This work represents a vital contribution to AHRQ’s ongoing efforts to combat both COVID-19 and the longstanding pandemic of antimicrobial-resistant infections.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY / ABSTRACT The combination of COVID-19 and alcohol/substance use disorders exacerbates a wide range of existing problems, including the likelihood of contracting COVID and severity of consequences, pandemic-related stresses that trigger alcohol and substance use, loss of jobs and healthcare access, increased interpersonal violence, and overarching systemic inequities. Interventions are needed to address these serious problems, which are likely to persist even after widespread availability of COVID vaccines. In response to PAR 20-243, this R01 project is a Hybrid II RCT/implementation study modifying and testing two of our alcohol smartphone interventions to address the fallout from COVID. We propose a 3-arm RCT comparing a control vs. a drinker-focused intervention vs. a family-focused intervention. The drinker-focused intervention (ACHESS-C) is an extension of our evidence-based Addiction–Comprehensive Health Enhancement Support system (ACHESS), augmented with COVID resources. The family-focused intervention (FamCHESS-C) combines ACHESS-C services with evidence-based Alcohol Behavioral Couple Therapy services to help both drinker and partner with behavior change, relationship problems, and general well-being. In the proposed 8-month trial plus 4-month follow-up, 198 dyads (drinker + family member) will be randomly assigned to: 1) Smartphone control: both receive a smartphone with standard support and crisis numbers; 2) ACHESS-C: drinker receives a phone with ACHESS-C, partner receives a phone with support and crisis numbers; 3) Fam-CHESS-C: both receive phone with FamCHESS-C. The project has the following aims: Aim 1: Complete refinements to the FamCHESS-C app. Aim 2: Conduct a balanced RCT to test the following outcomes: Primary: 1) drinker % heavy drinking days, 2) dyad quality of life. Secondary: 3) dyad relationship satisfaction, 4) dyad psychological/physical conflict, 5) drinker no heavy drinking days, 6) drinker % days alcohol/drug use, 7) dyad COVID vaccination rates, 8) drinker alcohol- and drug-related problems. Exploratory: 9) partner % days alcohol/drug use, 10) dyad crisis healthcare use, 11) dyad technology satisfaction. We hypothesize that outcomes will be more favorable in FamCHESS-C relative to ACHESS-C, and both will be more favorable relative to smartphone control. Aim 3: Examine mediation effects of dyad's competence, relatedness, and motivation; drinker's interim change in % days of alcohol and drug use, and extent of app use for comparisons of ACHESS-C and FamCHESS-C. Examine moderation of effects of condition by drinker sex, severity of drinker’s baseline alcohol use, drinker engagement in treatment for AUD/SUD, and dyad’s baseline relationship satisfaction. Aim 4: Conduct a small-scale (20 dyads) formative evaluation using an implementation science model to collect qualitative data on perceptions of difficulties and benefits of ACHESS-C and FamCHESS-C use.

NIH Research Projects · FY 2025 · 2021-09

While dietary sugars can alter the bacteriophage community in the gut ecosystem, the underlying mechanisms driving these changes remain elusive. Until we have filled these voids in our knowledge base, we will not be in a position to comprehend the interplay between dietary sugars, and probiotics and their viruses; this hampers the development of rational approaches to use diet to promote the efficacy of (engineered) probiotics. The long- term goals are (i) to unravel the mechanisms that drive the interplay between diet, and probiotic bacteria and their viruses, and (ii) to develop microbial therapeutics. The objectives of this research program are (1) to eluci- date the mechanisms by which sucrose promotes phage production in the probiotic gut symbiont Lactobacillus reuteri, and (2) to exploit diet-induced phage production to promote colonization and the release of therapeutics. The overarching hypothesis is that sucrose increases phage production, which consequently promotes coloni- zation and the release of recombinant proteins from engineered probiotics. The rationale of the work proposed is that its successful completion is expected to result in a paradigm shift of our understanding how diet impacts phage production, which will open up new and exciting avenues to modulate the gut microbiota composition, promote probiotic growth, and to tailor therapeutic delivery. The overarching hypothesis will be tested by pursuing three specific aims: (1) To characterize the sucrose metabolism pathway in relation to phage production; (2) To determine the role of phage on L. reuteri gut fitness in response to a diet enriched in sucrose; and (3) To use dietary sugar to control phage-mediated lysis and therapeutic delivery. In the first aim, targeted mutagenesis is used to dissect the sucrose metabolism pathway and their products to determine the triggers of sucrose-driven phage production in L. reuteri. Under the second aim, bacterial competition assays in gnotobiotic mice will de- termine the ecological ramifications of sucrose-driven phage production; isogenic mutants with reduced ability to metabolize sucrose, and to produce phage, are expected to reveal causation. Under the third aim, L. reuteri will be engineered to lyse—and release recombinant interleukin-22—in response to metabolism of a specific sugar. In an animal model of fatty liver disease therapeutic efficacy in response to diet will be investigated. This research is innovative because an important mutualistic gut symbiont is used to unravel the mechanisms by which sucrose boosts phage production in the gut ecosystem, which can be applied towards the development of next-generation probiotics. The research is significant because understanding how a dietary sugar boosts phage production in the gut ecosystem, and what the ecological ramifications are, opens up previously unexplored opportunities to alter the composition of the gut microbiota, which may include enrichment and/or engraftment of probiotic bacteria. Also, after we have demonstrated that a dietary sugar promotes the release of a recombinant therapeutic protein, a foundation is created for a realistic prospective to regulate therapeutic release in the gut using dietary or environmental triggers that activate phage-mediated lysis.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) results from autoimmune-mediated destruction of pancreatic β-cells. Despite its autoimmune etiology, emerging data suggest that intrinsic β-cell stress and defective adaptive stress responses can play an important role in the loss of functional β-cell mass in T1D. However, the molecular mechanisms by which the stress responses regulate β-cell death/survival in T1D have remained elusive, due primarily to a lack of in vivo preclinical genetic models, hindering the development of novel, effective, and alternative therapeutic strategies against T1D. Endoplasmic reticulum (ER) stress is caused by protein misfolding, chronic inflammation, and environmental factors. Upon ER stress, the unfolded protein response (UPR), a signaling cascade mediated by ER membrane-localized sensors ATF6, IRE1α and PERK, is triggered to re-establish cellular homeostasis. While these proteins induce adaptive responses under acute stress, under prolonged stress the UPR initiates apoptosis. The decision mechanisms for switching between adaptive and maladaptive responses, and the specific adaptive or maladaptive functions of each UPR sensors in distinct cell types and disease contexts, are yet to be uncovered. To this end, we have recently deleted Atf6 in β-cells (Atf6β-/-) of a well-established preclinical T1D model, non-obese diabetes (NOD) mice, before the initiation of islet inflammation. Remarkably, Atf6β-/- mice exhibited significantly reduced diabetes incidence . Transcriptome analysis of sorted β-cells of NOD Atf6β-/- mice revealed p53/p21 signaling pathway as the top enriched pathway and uncovered a previously not recognized pro-survival adaptative program in β-cells during T1D progression, which ultimately confers protection from T1D. Atf6β-/- mice also showed reduced insulitis and increased expression of immune inhibitory markers in β- cells, suggesting a non-cell autonomous effect of loss of function of Atf6 on the immune system. Therefore, in light of these data we hypothesize that upon loss of Atf6 in β-cells, a novel adaptive program governed by p21 signaling prevails, which in a non-cell autonomous manner alters β-cells-immune cell communication. Moreover, we hypothesize that under acute versus mild and prolonged stress conditions ATF6 triggers distinct transcriptional programs to regulate cellular homeostasis in human β-cells. Here, by utilizing a mouse model and human islets combined with a comprehensive toolbox of techniques and novel reagents we propose to (i) identify the mechanisms, by which loss of Atf6 in b-cells impact, b-cell-immune cell crosstalk (ii) define the mechanisms of p21 upregulation and reduced pathology in Atf6β-/- mice, and (iii) determine the ATF6-mediated stress adaptation mechanisms in human islets exposed to acute and prolonged ER stress. The successful completion of these studies will fill an existing gap in our knowledge base regarding the function of Atf6 in β-cells, identify a novel mechanism for β-cell-immune cell crosstalk, and significantly improve our understanding of mechanisms of β- cell failure in T1D. It will also provide mechanistic insight for future studies and support alternative translational strategies for T1D that target the b-cell UPR.