Mayo Clinic Rochester

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer-related deaths in the USA and is anticipated to become the second leading cause by 2030. Its characteristic desmoplastic stroma, constituting 60–70% of its volume, is one of the critical factors that contributes to the dismal outcomes. Fibroblast activation protein (FAP)-expressing Cancer-Associated Fibroblasts (CAFs) are one of the most important stromal constituents because they play a fundamental role in the carcinogenesis, fibrosis, tumor growth, metastases, and treatment resistance. FAP expression in PDA is an independent predictor of poor outcomes. Lack of noninvasive tools to precisely profile CAF identity and function both temporally and spatially in vivo is a critical barrier for translation of existing knowledge of the tumor microenvironment to address unmet clinical needs. 68Ga-FAP-inhibitor (FAPI)-46 has emerged as a PET radiotracer with optimal properties for FAP-targeted clinical imaging and theranostics in PDA. These include low nanomolar affinity to FAP, near-complete internalization of radioactivity bound to FAP, absence of physiologic uptake, rapid blood clearance and prolonged tumor retention, and operational characteristics that offer tremendous flexibility to suit the clinical context, PET scanner profile, and workflow for patients with PDA. Traditionally, long regulatory and reimbursement approval pathways coupled with high costs of comparative studies have delayed clinical access to promising precision tools such as 68Ga- FAPI-46 PET. Thus, for the clinical translation of a theranostic radiotracer such as 68Ga-FAPI-46, an academic- industrial partnership (AIP) based on complementary strengths and a coherent clinical development strategy is needed to reduce the risk and raise the likelihood of meeting FDA standards and consumer expectations. Our AIP - Mayo Clinic and Sofie Biosciences (“SOFIE”) - will undertake a clinical investigation in compliance with FDA standards to form the basis of a new drug application (NDA) with the goal to deliver a new capability to end users, consistent with this FOA’s intent. Our hypothesis is that 68Ga-FAPI-46 PET will be an accurate technique to detect and quantify CAFs and that metrics derived from 68Ga-FAPI-46 PET will be novel biomarkers in PDA. In Aim 1, using immunophenotyping as the reference standard, the sensitivity and specificity of 68Ga-FAPI-46 PET will be evaluated for the detection and quantification of CAFs in PDA, along with inter-reader and intra- reader reliability, and the dynamic changes in 68Ga-FAPI-46 PET biomarkers in response to neoadjuvant treatment. In Aim 2, 68Ga-FAPI-46 PET will be compared, correlated, and combined with other mechanistically distinct investigations to improve pre-surgical staging and to predict post-surgical outcomes. Our AIP has the potential to deliver a noninvasive molecular imaging assay that can provide greater insight into disease biology, impact clinical practice, predict outcomes, potentiate existing therapeutics, and yield a pathway to novel therapeutic approaches. Given the wide and evolving role of FAPI imaging and theranostics, our AIP has the potential to scale our impact beyond PDA to other oncologic and non-oncologic applications.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, partially owing to lack of effective biomarkers and/or screening strategies. The 5-year survival of patients with localized PDAC is 39% compared to 3% in patients diagnosed with metastatic disease, which indicates that detecting PDAC at early stage positively impacts survival. A centrally curated/managed multi-institution biospecimen resource containing prospectively collected, well-annotated biospecimens representing clinically relevant, diverse PDAC screening populations is desperately needed. The overall goal of the Pancreatic Cancer Detection Consortium (PCDC) Management and Data Coordinating Center (MDCU) is to facilitate the PCDC’s role in developing and utilizing high quality, well annotated samples for PDAC biomarker discovery, triage, pre-validation, and validation. The PCDC consortium is composed of multiple U01 Research Units (RU) and the MDCU, with NCI cooperation and central biospecimen storage at the NCI Frederick Central Repository. We will leverage our expertise and knowledge gained during the previous grant cycle to reinforce existing and new PCDC initiatives through expanded coordination, and strengthening the infrastructure to support management of the PCDC Reference Sets and Signature Protocols. Our Specific Aims are 1) To provide outstanding and timely administrative coordination and logistical support for the Pancreatic Cancer Detection Consortium. 2) To strategically plan, coordinate and implement the development of a well-annotated, uniformly collected and managed central PCDC biorepository and database. 3) To provide the highest quality biostatistical leadership of PCDC collaborative experimental design, study conduct, and analysis, and to provide biostatistical consultation to PCDC RUs. The strengths of the MDCU are the breadth and depth of knowledge and understanding of PDAC provided by MDCU personnel who will innovatively apply time tested infrastructure tools and strategies to consortium management, and our vast experience in enhancing the scientific needs of PCDC collaborative research. Our multidisciplinary team is committed to continue its leadership and contribution to the PCDC organization to advance the early detection of pancreatic cancer.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY PD is a complex neurodegenerative disease with a broad spectrum of motor and non-motor features. The cardinal motor features include resting tremor, rigidity, bradykinesia (or akinesia), and postural instability. Over the past decade, deep brain stimulation (DBS) has largely replaced ablative techniques in the surgical Although clinical phenotypes of PD such as tremor dominant (TD) and postural instability and gait difficulty (PIGD) subtypes have been identified since the mid-1990s, to our knowledge, no neurobiomarkers have been identified to describe them and assist with decision-making regarding the DBS therapeutic target. treatment of PD. Very limited data exists regarding the electrophysiological abnormalities within the basal ganglia and associated structures which likely accompany the symptom severity or the phenotypic subtypes of PD. This proposal seeks to overcome these limitations by investigating the spatio-spectral dynamics of local field potentials (LFPs) and single unit activity recorded from the subthalamic nucleus (STN) and globus pallidus internus (GPi) and relate them to symptom manifestations, in an attempt to define the differences in PD motor phenotypes. In our preliminary studies and to the best of our knowledge, we are the first to show initial evidence that high frequency oscillations of microelectrode LFPs and their nonlinear interaction with the beta band in the form of phase amplitude coupling can distinguish PD motor phenotypes in the territories of STN. Based on our preliminary observations, this project will investigate these phenotypic LFP patterns in large patient populations in STN and GPi. Specifically, by employing machine learning techniques, neural signatures in STN and GPi will be discovered to differentiate motor phenotypes in PD. The project will investigate to what extent the extracted neural patterns can serve as objective neurobiomarkers to identify the territories of basal ganglia causing symptom differences. In addition, retrospectively, the project will also explore to what extent localization of these phenotypic LFP patterns with directional macro electrodes can describe the efficacy of chronic DBS. If successful, these advancements will provide unique opportunities to understand symptom manifestation and personalization of DBS in patients with PD.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT This K23 application outlines a career development plan that will advance Dr. Heidi Lindroth on her pathway to becoming an extramurally funded, independent clinician-nurse scientist. Her long-term career goal is to become an international leader focused on mitigating delirium severity in older adults, reducing the risk and burden of adverse outcomes, including Alzheimer’s Disease and related Dementias (ADRD). Delirium, an acute and fluctuating disturbance in consciousness and cognition, impacts 70-80% of mechanically ventilated, intensive care unit (ICU) older adults. Severe delirium accelerates the rate of global cognitive decline leading to an increased risk of ADRD. Therefore, reducing delirium severity holds great promise in decreasing ADRD burden. Unfortunately, delirium severity is seldom monitored in the ICU. Accurate and timely measurement of delirium severity is urgently needed to implement the proper, evidence-based treatment at the right time. To begin to fill this significant gap in clinical care, Dr. Lindroth’s proposed research study will leverage the foundational work completed in the Mayo Clinic Herasevich Clinical Informatics Laboratory to develop and preliminary test a passive digital marker (PDM) for delirium severity in critically ill, older adults. Like a continuous vital sign, this PDM would provide immediate and actionable feedback to clinicians on the status of delirium severity in ICU patients. Older adult (>65 years old) patients who are anticipated to remain in the ICU for >24 hours will be prospectively recruited upon their admission to the medical ICU at Mayo Clinic, Rochester, MN, and the general ICU at the Mayo Clinic, Eau Claire, WI for both stages of the study (development-aims 1 and 2; pilot RCT-aim 3; total n=230) and followed until either death and/or discharge from the ICU. Data collected will include continuous digital video recordings of the patient in their ICU room, routine EHR data, and study team administered delirium severity assessments. The pilot RCT in Aim 3 will randomize participants (1:1, computer-generated assignment) to either the intervention (PDM for delirium severity) or usual care with the primary outcomes of usability and acceptability. Dr. Lindroth has co-designed a career development plan and a research proposal with her transdisciplinary mentorship team to accomplish the following short-term objectives to: 1) acquire informatics and data science knowledge to design/build tools that support clinical decision making; 2) gain experience in the conduct of prospective ICU clinical trials in older adults, and; 3) acquire and develop grant writing skills. These short-term objectives will be accomplished through formal training (Masters in Artificial Intelligence in Healthcare), experiential learning (conduct of proposed study), and focused mentorship Thus, this K23 award will allow Dr. Lindroth to progress in her career development plan and provide her with necessary protected time to acquire the outlined research skills, knowledge, and experience to continue her pathway towards independence.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Enteric neurons (ENs) are required to control gastrointestinal (GI) motility by regulating neurotransmission. Loss of ENs has been demonstrated in digestive diseases in adults and during aging. In the muscularis propria of the GI tract, diverse populations of macrophages, called muscularis macrophages (MMs), are linked to the normal development and maintenance of ENs. While we have shown that MMs closely interact with ENs, the molecules that may regulate MMs-ENs functional interactions are unknown. Thus, to use MMs as a target for regulating ENs in digestive diseases, we first need to characterize MMs’ phenotype in humans and identify the mechanisms regulating MMs-ENs interaction. Our long-term goal is to determine the signaling pathways regulating MMs-ENs functional interaction and use this knowledge to develop new therapeutic strategies to treat digestive diseases. In preliminary data generated for this application, we discovered (1) a new population of human MMs closely associated with nerve fibers. (2) Depletion of this newly discovered MMs population from human organotypic cultures of small intestine muscularis propria reduces nerve fibers. (3) RNAs from tissues of patients with slow transit constipation (STC) have reduced expression of Complement 1qa (C1qa), one of the genes enriched in the newly discovered population of MMs. (4) C1qa, one of the genes enriched in the newly discovered human MMs population, is exclusively expressed by MMs in mice. (5) Depleting C1qa from MMs reduces synaptic marker expression, alters GI contractility, and reduces whole gut transit time. (6) Conditional depletion of CSF1 from ENs in a transgenic mouse model induces loss of the newly discovered population of MMs, ENs and reduces whole gut transit time. Thus, the central hypothesis of this application is that C1qa expressing MMs regulate GI neurotransmission, and their phenotype depends on CSF1 released from closely associated ENs. In SA1, we plan to characterize this newly discovered MMs population in STC patients and assess its contribution to GI contractility using human organotypic cultures. In SA2, we plan to study the contribution of this novel population of MMs to neurotransmission. In SA3, we propose to study the role of EN-released CSF1 on circulating monocytes’ recruitment and differentiation into the novel population of MMs. In the long term, we expect these studies will contribute substantially to creating the basis for targeting MMs as a novel therapeutic approach to regulate ENs in digestive diseases, such as STC, characterized by loss of ENs.

NIH Research Projects · FY 2025 · 2022-09

Summary/Abstract Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with dismal survival for patients with unresectable disease. Recent approval for PD-L1 inhibitor atezolizumab (atezo) and VEGF inhibitor bevacizumab (bev) in frontline treatment is a landmark advance; however further improvement is needed with a median progression free survival of less than 7 months. We have found through a multi-center cooperative study that tumor transcriptome signature high in interferon-gamma and MHC-II signaling (INFAP signature) is correlated with increased response and survival to PD-1 inhibitor. We propose to add high dose external beam radiotherapy (EBRT) followed by intra-tumor injection of autologous dendritic cells (DC) to atezo/bev to further enhance the immune stimulatory effect. Radiation can induce inflammatory tumor cell death that can be favorable for tumor neoantigen presentation. Injection of autologous DC after EBRT would be a novel method of boosting in vivo tumor antigen uptake and presentation to expand tumor-reactive cytotoxic T cells. We have treated subjects with unresectable liver tumors (HCC and cholangiocarcinoma) in a pilot study with this EBRT and DC approach with promising response and acceptable toxicity (no grade ≥3 toxicity). Three of the five subjects had partial response, including a patient with ongoing response beyond 1 year. Both emergence of new TCR clones and expansion of existing TCR clones, including clones with tumor reactive and cytotoxic profile, have been observed, suggesting this combination could enhance tumor reactive cytotoxic T cell response. However, many of the TCR clones also have early exhaustion signal with upregulation of multiple checkpoint receptors. Thus, combining DC injection with atezo/bev may help further enhance the cytotoxic functions of these TCR clones. We hypothesize that combining EBRT followed by intratumor DC injections with atezolizumab and bevacizumab can improve the PFS for patients with unresectable HCC and that the effect is mediated by systemic expansion of a tumor reactive T cell repertoire. We will test the hypothesis through 2 aims. 1) Assess the clinical efficacy of this combination therapy in a phase II study with a safety run-in phase. Increased PFS rate at 1 year will be the primary endpoint. 2) identify the effect of this novel combination immunotherapy on tumor reactive T cell repertoire. We will use scRNAseq and TCRseq to identify TCR clonal expansion and transcriptome profile of the TCR clones in the blood and tumor, with a focus on tumor reactive TCR clones. We will also use scRNAseq and flow to profile the changes of other immune cells in the tumor and blood, with a focus on the changes in expression of the INFAP signature. Finally, we will use imaging cytometry to examine the tumor and immune spatial relationship in the tumor. Our study will not only identify the clinical activities of this novel combination therapy but also use state-of-the-art technology to improve our understanding on the mechanism of action to this immunotherapy.

NIH Research Projects · FY 2025 · 2022-09

Translation of a novel combination therapy approach for non-Hodgkin lymphoma . Diffuse large B-cell lymphoma is the most common type of Non-hodgkin’s lymphoma (NHL) with limited treatment options in the relapsed or refractory (r/r) setting. This is true in humans and dogs. Immunotherapy with checkpoint inhibitors (CPIs) have demonstrated durable efficacy for Hodgkin’s lymphoma, but poor efficacy for NHL. There is an unmet clinical understand mechanisms of immunotherapy resistance and develop therapeutic approaches to improve clinical response for patients with advanced NHL and other cancers. DLBCL in companion dogs (cDLBCL) is treated with similar chemotherapy protocols and has a similarly poor prognosis in the r/r setting as human DLBCL. While genomic comparison shows limited overlap of the mutational landscape in canine and human DLBCLs, preliminary comparison of the tumor microenvironment (TME) shows conservation of stromal and immune compartments between the two species. Thus, cDLBLCL provide opportunities to prospectively investigate clinical toxicities and mechanisms of clinical response in a clinically realistic setting that recapitulates the pathology, heterogeneity, and TME of human cancers. Vesicular stomatitis virus (VSV) is a rapidly replicating, robustly immunogenic oncolytic virus (OV) platform that has been engineered for safe systemic therapy of disseminated cancer. Intravenous (IV) VSV therapy was shown preclinically in murine tumor models to rapidly infect, spread within, and kill tumor cells, and induce robust intratumoral immune infiltration, sensitizing tumors to checkpoint blockade. ONIx (oncoimmunology accelerator) is a novel, dual targeted CPI that targets both innate and adaptive mechanisms of tumor immune suppression to enhance antitumor immune responses mediated by macrophages and T-cells. We hypothesize that oncolytic VSV and ONIx will have complementary mechanisms of action (MOA), working in concert to kill tumor cells by direct viral lysis as well as phagocytosis, increase availability of tumor associated antigens (TAAs), promote antigen presentation and activate anti-tumor T-cell responses, to enhance immune mediated tumor killing and improve clinical responses in r/r DLBCL. Our proposal merges the expertise and resources of leading institutions in OV development (Mayo Clinic), comparative oncology (University of Minnesota), and lymphoma immunotherapy (Mayo Lymphoma SPORE) to perform a veterinary trial and correlative studies to evaluate the safety and preliminary efficacy of this novel combination therapy in r/r cDLBCL. The proposed studies will yield valuable insights into how an IV administered OV can infect heterogeneous DLBCL tumors and agitate the TME; if this disruption enhances the ability of CPIs (and potentially other immunotherapies) to activate immune mediated tumor killing; and how the tumor architecture differs in the context of clinical response versus non-response. The heterogeneity inherent in naturally occurring cDLBCL will inform the clinical utility of this combination therapy, define MOA, and identify biomarkers that can be explored clinically in human DLBCL.

NIH Research Projects · FY 2025 · 2022-08

Idiopathic pulmonary fibrosis (IPF) is a common form of interstitial lung disease (ILD), resulting in alveolar remodeling and progressive loss of pulmonary function, respiratory failure, and death often within 5 years of diagnosis. Genetic and experimental evidence support the concept that chronic alveolar epithelial injury and failure to properly repair the respiratory epithelium are intrinsic to IPF disease pathogenesis. Histologically, respiratory epithelial cells in the lung parenchyma are replaced by cells which are normally restricted to conducting airways. Fibrotic lesions and honeycomb structures replace alveoli, the latter normally lined by alveolar type 1 (AT1) and AT2 cells. Acute exacerbations by respiratory viral infections are the most devastating complication of IPF, having an in-hospital mortality rate of greater than 50%. Data from previous coronavirus pandemics such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), as well as emerging data from the COVID-19 pandemic, suggest there could be substantial fibrotic consequences following SARS-CoV-2 infection, the causative agent of COVID-19. Interestingly, the major risk factors for severe COVID-19 are shared with idiopathic pulmonary fibrosis (IPF), namely increasing age, male sex, and comorbidities such as hypertension and diabetes. Although many patients who develop acute respiratory distress syndrome (ARDS) survive the acute phase of the illness, a substantial proportion die as a result of progressive pulmonary fibrosis. It remains unclear why certain individuals are able to recover from ARDS, whereas in others there is a shift to unchecked cellular proliferation with the accumulation of BC-pods, fibroblasts and myofibroblasts. In these patients, there is also excessive deposition of collagen alongside other components of the extracellular matrix resulting in progressive pulmonary fibrosis. Distinct epithelial stem/progenitor cell pools and/or their mesenchymal niches repopulate injured tissue depending on the extent and type of injury, and the outcomes of regeneration or fibrosis in response to severe alveolar epithelial injury is dependent in part on the dynamics of cell competition between these cell populations. In tissues harboring a mosaic imbalance in cMyc or Yap protein levels, cells with higher cMyc or nuclear Yap levels become super- competitors and expand at the expense of cells with lower levels, by eliminating them. Alternatively, if certain stem cell populations are selectively wiped out due to the type of injury, other stem cell populations that escape the injury and which may not be so adept at replacing the destroyed tissue will now have a competitive advantage. For example, SARS-CoV-2 enters respiratory epithelial cells via its receptor, angiotensin-converting enzyme 2 (ACE2), causing severe airway and alveolar epithelial injury. Based on Ace2 expression, distinct stem/progenitor cell pools appear to be differentially susceptible to SARS-CoV-2 infection. This grant proposal seeks to manipulate the underlying mechanisms of cell competition to help prevent and treat IPF and ARDS. Cell competition might also be exploited to maximize the potential of healthy tissue replacement.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Cells have evolved complex machinery for both the replication of DNA and for repairing errors in DNA. Herpes Simplex Virus 1 (HSV) is a large double strand DNA virus that replicates in the nucleus of the host cell and commandeers some of this host replication machinery. Despite decades of study, the mechanisms of HSV DNA replication are still poorly understood. The incoming genome contains nicks and gaps, and it is not known when or if these are repaired in relation to the timing of DNA synthesis. HSV encodes seven essential DNA replication proteins including an origin binding protein, a single strand DNA (ssDNA) binding protein (ICP8), a three- subunit helicase/primase (UL5/UL8/UL52), and a two-subunit polymerase (UL30/UL42). In addition, HSV also encodes a two-unit recombinase consisting of a 5'-3' exonuclease (UL12) that functions with ICP8. Isolation of proteins on nascent DNA (iPOND) is a powerful tool to study DNA replication because it allows for the specific purification of replication forks away from bulk chromatin. When coupled with SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics, the iPOND-SILAC-MS method provides a robust, unbiased discovery tool to identify fork associated proteins by determining the intensity of proteins in a pulse sample compared to a chase sample. We have utilized iPOND-SILAC-MS to generate robust data sets of the protein composition of HSV replication forks and replication forks lacking UL12. In the absence of UL12 a cellular deubiquitinating enzyme, USP15, is not recruited to replication forks. USP15 interacts directly with UL12 and is required for efficient HSV replication. In addition to viral proteins, many cellular proteins are enriched on viral replication forks. In twelve iPOND-SILAC- MS we have identified 200-300 proteins (viral and host) enriched on HSV DNA replication forks. The overall goal of this research proposal is to identify the host proteins associated with viral DNA to generate a more complete understanding of the mechanisms of HSV DNA replication. To this end we will address three overlapping research areas in my laboratory: 1) Characterize the physical interaction of UL12 with USP15, 2) Identify the human replisome proteins required for HSV DNA replication, 3) Determine the fate of the nicks and gaps in the incoming viral genome.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Individuals with germline variants in breast, ovarian and pancreatic cancer predisposition genes are at significantly elevated risk of developing these cancers in their lifetime. Clinical hereditary cancer genetic testing for pathogenic variants in these genes has become an important part of clinical practice. Much of the benefits of genetic testing are associated with the BRCA1 and BRCA2 genes because of the risk management, surgical prevention and targeted treatment benefits associated with knowledge of the presence of a cancer predisposing pathogenic variants. However, identification of pathogenic variants in other predisposition genes including ATM, BARD1, BRIP1, CHEK2, PALB2, RAD51C and RAD51D is also clincially meaningful because carriers may qualify for enhanced screening for breast, ovarian and pancreatic cancer. However, this process is often complicated by an inability to establish the clinical relevance of variants in these genes. This lack of information about these variants means that individuals carrying germline variants often cannot benefit from enhanced risk assessment and management or make informed decisions about surgical prevention or tailored treatment options. To address this issue, we developed the BRCA1/2 and Hereditary Breast, Ovarian, and Pancreatic (HBOP) Variant Curation Expert Panels (VCEPs). We will build upon our initial work to implement ClinGen rules- based methods for variant classification in BRCA1, BRCA2, ATM and PALB2 and also develop and implement rules for curation of variants in CHEK2, RAD51C, RAD51D, BARD1 and BRIP1. Thus, the goal of this application is to curate and classify the clinical relevance of germline variants in BRCA1 and BRCA2 through a BRCA1/2 VCEP and variants in ATM, BARD1, BRIP1, CHEK2, PALB2, RAD51C and RAD51D through the HBOP VCEP. The results from the proposed curation efforts will be entered into the ClinGen Variant Curation Interface and made available to the public through the ClinVar and BRCA Exchange websites.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY The goal of this project is to generate a novel technological pipeline to phenotype senescent cell identity (defined by biomarkers, function-related transcriptional profiles, morphology, and microenvironment) and composition (defined by quantity, diversity, and distribution) across any mouse or human tissue. Use of diverse profiling methods has revealed that senescent cells are highly heterogeneous and adversely influence tissue health and function. However, due to biological heterogeneity and reliance on diverse methods, we do not comprehensively understand the identities of senescent cells and extent to which they contribute to age-related decline. This limits the ability to devise effective therapeutics that could have important societal benefit. To fill these significant knowledge gaps, we require new technologies that accurately characterize heterogenous senescent cell states in aged tissues. We will pioneer iterative and integrated use of imaging mass cytometry (IMC) and transcriptomic digital spatial profiling (DSP) to molecularly phenotype senescent cells in aged tissues. Our preliminary data generated from high-dimensional spatially-resolved and suspension-based mapping technologies demonstrate that microglia, neurons, and additional cell types display distinct senescent profiles in the aged mouse brain. Unique properties of the brain include substantial cell and regional heterogeneity, limited regenerative capacity, age-vulnerability, and pleiotropic presentation of senescence-related biomarkers, all within well-defined micro- environments. These features support an initial experimental focus on the brain for feasible and comprehensive resolution of the anticipated panoply of senescent identities and contexts. Thus, we will develop this novel tech- nology to map senescent cells, first, in the hippocampus and cortex of aged mice (UG3 phase) and subsequently, across brain regions in female and male mice throughout the lifespan (UH3 phase). Critically, our innovative technological pipeline, by design, will be broadly applicable to any tissues through customization of cell-identity and senescence biomarkers. We will leverage experience studying cell senescence and the SASP in diverse tissues to collaboratively adapt and scale IMC and DSP to generate mouse and human senescent cell atlases. High-dimensional multimarker imaging and spatial transcriptomics are anticipated to revolutionize the ability to rigorously and comprehensively characterize and map senescent cells in distinct tissue contexts. Ultimately, use of this novel technology may fundamentally advance understanding of how cell senescence contributes to age- related tissue dysfunction and may reveal new strategies to disrupt senescence-mediated pathology.

NIH Research Projects · FY 2026 · 2022-08

Project Summary Type 1 diabetes (T1D) is strongly associated with coronary heart disease. However, the molecular mechanism underlying coronary vascular pathology, especially coronary arterial smooth muscle pathology in human with T1D is incomplete. Vascular BK channels, composed of four pore-forming subunits (BK-α) and four regulatory subunits (BK-β1), are densely expressed in coronary artery smooth muscle cells (SMCs) and are a key determinant of coronary blood flow and cardiac function. Over the last 10 years, we and other investigators have demonstrated that coronary BK channel function is impaired in T1D animals due to increased oxidative stress and it contributes to a worse outcome in myocardial ischemia. However, most of our knowledge of coronary BK channel dysregulation in T1D is obtained from animals and most of studies are focused on the BK-β1 dysregulation in diabetes. The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is a component of cytoskeleton proteins in vascular SMCs. Sorbs2 is abundantly expressed in cardiovascular tissues and is a downstream target of Nrf2. However, the role of Sorbs2 in vascular pathophysiology is unknown. We have exciting preliminary results showing that Sorbs2 interacts with BK-α and BK-β1 protein and regulates BK channel expression in coronary SMCs. Interestingly, Sorbs2 knockout mice share many common features of coronary BK channelopathy with diabetes, despite being normoglycemic and not obese, indicating that Sorbs2 deficiency is an independent risk of vascular BK channelopathy. Importantly, Sorbs2 expression is significantly reduced in coronary arteries of patients with T1D. Unlike T2D patients, the expression of BK-α, but that of BK- β1, is markedly reduced in the coronary SMCs of patients with T1D. However, the role of Sorbs2 on coronary BK channelopathy and vasculopathy of human T1D has not been established, and the underlying mechanisms regarding the downregulation of BK-α expression in coronary SMCs of T1D patients is unclear. In this project, we will take advantage of the availability of human coronary arteries from T1D patients who are scheduled for cardiac surgery at Mayo Clinic in Rochester (MN) to test our hypothesis that downregulation of Sorbs2 expression contributes to BK channel and vascular dysfunction in the coronary arteries of T1D patients and increase of Sorbs2 expression by pharmacological Nrf2 activation protects coronary BK channel function and vasoreactivity in human tissues with T1D. Results from this study will provide novel insights into the molecular mechanisms underlying BK channelopathy and coronary vasculopathy in T1D and may help develop new strategies for the treatment of cardiovascular complications in T1D patients.

NIH Research Projects · FY 2025 · 2022-08

Unnecessary discharges from a hospital to a skilled nursing facility (SNF) are costly and may accelerate patients’ functional losses and requirement for long-term institutionalization. Patients with Alzheimer's Disease and Alzheimer's Disease Related Dementias (AD/ADRD) and other types of cognitive impairment are uniquely disadvantaged by this status quo in that they are twice as likely to be hospitalized, four times more likely to be discharged to SNFs with less than 50% returning to their homes. This situation can be addressed as it is the product of a typically rushed discharge planning process with inadequate time to discover, much less address, a patient’s barriers to home discharge. Recent reports suggest that as many as a third of patients dismissed to SNFs, including those with AD/ADRD, could return directly home if their post-acute care (PAC) needs and barriers were anticipated and addressed. Several key deficits prevent broad realization of a patients’ potential to discharge directly home, or their Home PAC Potential (HoPe). These include a limited ability to: 1) quantify factors that determine PAC needs, 2) identify and address remediable barriers to home discharge, and 3) mobilize stakeholders for advancement of individualized discharge plans. Collectively, these deficits prevent the timely initiation of acute care services that can realize a patient’s potential for home discharge, with PAC as necessary. Rehabilitation-focused, hospital-Home Healthcare Agency (HHA) partnerships have established that interdisciplinary care plans enacted early in a hospital stay with patient and caregiver involvement increase the likelihood of a patient’s return home. Our team developed an Epic electronic health record (EHR)-based discharge planning system that triangulates EHR, patient reported outcomes (PROs), and social determinants of health data to identify HoPe barriers and direct needs-matched rehabilitation service delivery. A pilot of the system among 358 patients increased the home discharge rate by over 25% and revealed high user acceptability. However, the pilot also identified the need to improve addressing of cognitive impairments, targeting of high-yield HoPe barriers, and engagement of non-clinical stakeholders. We propose to address these limitations by pursuing three Specific Aims: 1) Develop a low-burden computerized adaptive test PRO to assess the domains of functional cognition relevant to a safe home discharge; 2) Develop a machine learning algorithm to prioritize actionable HoPe barriers and estimate the degree of change needed for home discharge; and 3) Apply user-centered design principles to refine the EHR discharge planning system for optimal usability and enhanced EHR portal patient, caregiver, and HHA staff access. Our goal is to both integrate and pilot these deliverables in a mature and optimally usable EHR discharge planning system, and to evaluate the feasibility and acceptability of its implementation. We anticipate that the system will be scalable, and amenable to inter-institution transfer for testing in a multi-site pragmatic trial.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT This K24 application seeks to provide protected time for Dr. Elena Myasoedova to mentor trainees in patient- oriented research (POR) aiming at defining the long-term temporal relationship between systemic inflammation, cardiovascular risk factors and cardiovascular events on the risk of Alzheimer’s Disease and other related dementias (ADRD), using rheumatoid arthritis (RA) as a prototype. This award will enable the applicant to solidify and advance her current NIA-funded R01 research program in new directions and to expand her mentoring opportunities and mentoring experience. Dr. Myasoedova is a tenure-track Associate Professor at Mayo Clinic. She has a strong and ongoing record of high-quality POR, publication, and mentoring in research directly related to the proposed studies. The applicant has shown significant abilities in mentoring trainees at all career stages and will continue to benefit from the rich and robust resources, unique research infrastructure and a large pool of patients and mentees at Mayo Clinic. During the funding period, further development of the applicant will include: 1) Improvement in mentorship skills in POR; 2) Enhancement of knowledge in biostatistics for POR; 3) Development of a collaborative niche for POR in aging and ADRD in rheumatic diseases; and 4) Maintaining uninterrupted extramural funding. These goals will be achieved through a combination of seminars, meetings, courses, and collaboration with the Division of Epidemiology, the Kogod Center on Aging, the Rochester Epidemiology Project and the Mayo Clinic Study on Aging. Dr. Myasoedova will further integrate and advance these skills through continued mentorship of trainees within currently funded POR and through expansion of her ADRD research through the K24 mechanism. Research aims of the proposal include: 1) Examining the temporal association between cardiovascular risk factors, systemic inflammation and ADRD in RA; 2) Evaluating direct and cardiovascular-mediated effects of chronic inflammation on ADRD in RA; and 3) Defining the population attributable risk (PAR) of RA characteristics on incident mild cognitive impairment and ADRD, overall and by sex. The proposed studies will inform risk stratification of ADRD in a chronic inflammatory setting of RA overall and by sex as a key step for advancing the understanding of ADRD risk and developing prevention strategies in patients with RA and beyond. These aims are well-aligned with NIA’s Goal D to improve our understanding of the aging brain and ADRD, specifically defining the impact of inflammatory processes on the development of AD, and whether systemic risk factors such as obesity, diabetes, hypertension, and heart disease during midlife are associated with accelerated age-related cognitive decline and with increased risk for AD. Successful completion of this award will facilitate the development of the next generation of patient-oriented researchers in ADRD and will expand Dr. Myasoedova’s POR program to ensure continued mentoring opportunities into the future, aligning with the NIA’s mission to “Foster the development of research and clinician scientists in aging’.

NIH Research Projects · FY 2024 · 2022-08

ABSTRACT The importance of the enteric (ENS) and autonomic nervous systems (ANS) in maintaining intestinal health is made evident in Hirschsprung disease (HSCR), defined by the absence of the ENS in distal bowel and caused by a number of mutations (e.g., Ednrb). HSCR is managed with surgery to remove the ‘abnormal’ bowel, but many patients suffer from persistent functional and inflammatory bowel complications (e.g., Hirschsprung Associated Enterocolitis, HAEC), indicating that the ENS in proximal bowel is also affected by HSCR-related mutations despite appearing anatomically normal. Our lab generated a novel HSCR mouse model with Ednrb mutations that expresses GCaMP, a genetically-encoded calcium indicator, to measure synaptic connectivity in proximal bowel where the ENS is present. By using in vivo and ex vivo colon preparations that keep intrinsic and extrinsic nerve pathways intact, we have previously shown how ENS, ANS (sympathetic and parasympathetic), and sensory inputs normally influence colon function. Ednrb-/- (null)-GCaMP mice had specific changes in myenteric neuron activity (spontaneous and synaptically-evoked) in proximal colon at post-natal ages that likely contribute to the chronic issues in HSCR. Interestingly, age-matched Ednrb+/- (het)-GCaMP mice displayed a similar functional phenotype, despite having ENS innervation in the entire bowel, and they continued to be significantly different from wild-type littermates as adults. These mice exhibited dysmotility, had altered immune cell counts in the lamina propria, and appeared to be more vulnerable to experimentally-induced inflammation. Therefore, ‘subclinical’ mutations in HSCR-related genes (i.e., those that would not cause a HSCR diagnosis) appear to be sufficient to impair aspects of ENS circuit development. Importantly, Ednrb-/- and Ednrb+/- GCaMP mice model different aspects of HSCR: ‘clinical’ HSCR complications in newborns and ‘subclinical’ HSCR issues that persist into childhood and adulthood. Several recent HSCR studies have identified shifts in the microbiome and impaired immunity that may underlie increased inflammation and HAEC, but the causal role of the microbiome in producing HSCR-related dysfunction is unknown. Based on what is known about neuro- immune-microbiota interactions, I hypothesize that the ENS/ANS contributes to immune dysfunction indirectly via microbiome changes due to dysmotility, but also potentially through changes in direct neuro-immune communication. To test this hypothesis, experiments in Aim 1 (K99) will define the immune cell profile and microbiome in ‘clinical’ and ‘subclinical’ HSCR mouse models at post-natal ages, and determine whether the ‘HSCR microbiome’ is necessary and/or sufficient to produce ENS, ANS, or immune dysfunction. Experiments in Aim 2 (R00) will test whether HSCR-related dysfunction in the ENS/ANS impairs the ability to respond to inflammatory immune challenge, and determine the role of the ‘HSCR microbiome’ in resolving inflammation. As numerous digestive disorders exhibit neural, microbiome and/or immune dysregulation, the proposed research program will broadly impact human health.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY The interaction between microbes and the immune system plays a key role in human health. Microbes can help train and develop major components of the host’s innate and adaptive immunity, while the immune system orchestrates the host-microbe symbiosis. Many studies use animal models, population level 16S rRNA gene and metagenomic sequencing to gain evidence on microbe-immune correlation, however, the underlying mechanism is relatively undefined due to the complexity of this ecosystem and the limitations of the available tools. The overarching goal of my research program is to understand how the biological functions of the microbial and cells of the innate immune system co-evolve. Dissecting the crosstalk between the microbial and immune cells is challenging and requires more sophisticated methods. My overall goal is to develop tools to understand, for example, how can microbial cells influence the innate immune system even if their number is few? How do the microbes and the immune system co-evolve according to the constantly changing strategies of the other? To probe these questions, I propose to use a bottom-up approach that starts a single microbial and immune cell and builds up complexity. Over the past 5 years, I developed a microfluidic platform and methods for single cell whole genome and transcriptome sequencing, suitable for the sequencing of both bacterial and human cells. This platform led to the discovery of preferential genetic alterations in microbes that adapted to extreme living conditions, and that the gene expression profile in individual microbial cells is distinct. During the next 5 years, I plan to modify and optimize this platform to investigate the bi-directional relationship between the microbial and immune cells. This work will be centered on dendritic cells and Staphylococcus aureus as an exemplary study, and then incorporate other cell types and environmental factors. Briefly, I plan to study how a single microbial cell can be perceived as a group to regulate the signaling pathways of a single immune cell through analyzing their transcriptional profiles. Central to this work is to integrate additional components into the platform to enable the co-culture of a single microbial and immune cell, activate and monitoring their signaling, and investigate the signaling pathways through whole transcriptome sequencing. This work will ultimately help map different immune cell phenotypes and their responses to various microbes, which will lead to the better understanding of the heterogenous and dynamic immune responses. In the long run, this research program will accelerate the study of how microbiome and the immune system are cross-regulated in more complex settings. Besides, these fundamental processes involve general biology principles at single cell transcriptional levels and are applicable to diverse host cells, lending broader significance to the proposed work.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Inappropriate use of thyroid ultrasound (iTUS) is an important driver of thyroid cancer overdiagnosis and overtreatment, which involves high-risk procedures and long-term therapeutics that cause medical, psychosocial, and financial hardships for patients. Cumulative annual cost of well-differentiated thyroid cancer care in the U.S. has been estimated to exceed $1.5 billion and is projected to reach $3.5 billion by 2030, and the potential cost after 5 years of thyroid cancer diagnosis is $50,000 per patient. Thyroid cancer is one of the fastest-growing cancers in the U.S, but mortality remains very low. Approximately 25% of new cases are attributable to the identification of small thyroid cancers that are unlikely to cause harm if they were left undiagnosed and untreated. The biggest driver of small thyroid cancer diagnosis is iTUS use in asymptomatic people, a practice discouraged by clinical guidelines. The pervasiveness of iTUS despite recommendations against it suggests the need for active strategies to eliminate it. The process of eliminating practices that are not evidence-based is known as de-implementation. To date, no studies have provided a replicable and useful way for health systems to identify their iTUS practices, and there has been no systematic evaluation of multilevel factors driving it, such that we lack key information about targeted, acceptable, and feasible de- implementation strategies. Without them, overuse will persist. To fill this gap, we will leverage a multidisciplinary team with vast experience in computer phenotyping expertise, machine learning, and mixed method research. We will also use two unique databases: the Rochester Epidemiology Project, a medical record-linkage system that captures health care information from the entire population of 27 counties in Minnesota and Wisconsin, and the Patient-Centered Clinical Research Network (PCORnet) that shares a common data model to organize data into a standard structure. There are three aims. Aim 1: Using the REP and two PCORnet sites, to develop a replicable computer phenotype to identify patients receiving iTUS. Aim 2: Using 4 PCORnet sities, to identify patient, clinician, and practice factors associated with iTUS in a representative sample of healthcare practices. Aim 3: Using mixed methods, to understand factors and identify potential strategies for iTUS de-implementation acceptable to the patient, clinician, and health system stakeholders. This proposal is responsive to the objectives of NOT-CA-20-021 to explore de-implementation of ineffective or low-value clinical practices along the cancer care continuum. At the end of this study, we will have developed and validated a computer phenotype to identify iTUS across diverse settings, as well as a list of acceptable strategies likely to decrease iTUS. These findings will be broadly disseminable and will pave the way for studies—deployed in diverse health systems and targeting patients, clincians, and organizations—that test the effectiveness of the de-implementation strategies identified here.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY / ABSTRACT Prostate cancer (PCa) is the most common and second most deadly non-cutaneous cancer in men in the USA, but five-year survival rates approaching 100% are possible if detected early. Prostate MRI has become increasingly important in cancer detection and localization. Two-dimensional (2D) multislice T2-weighted spin-echo (T2SE) and diffusion-weighted (DWI) acquisitions are mainstays of clinical prostate MRI, but radi- ological interpretation is hindered because through-plane resolution is 3–8× coarser than inplane resolution. This is particularly limiting in the transition zone (TZ) of the prostate where differences in relaxation times between malignant and normal tissue are small, and interpretation is often based on sharpness-related criteria such as encapsulation. Per PI-RADSv2.1, T2SE is the critical sequence. Reported sensitivity is far less than ideal, 30 to 70%. In addition to the specific need for improved diagnostic accuracy in the TZ, there is an initiative to make prostate MRI exams shorter and more efficient. Thus, there is a need to reconcile these competing demands of improved resolution and contrast via reduced partial volume effects with higher effi- ciency in prostate MRI. We have developed a super resolution (SR) method for T2SE which uses overlapped, standard-thickness (3mm) axial slices to form 1mm slices with reconstruction in kZ-space, dubbed kZ-multislice (KZM). We have shown in vivo the superior sharpness of the resultant images vs. 3mm slices for equal inplane resolution. More recently we have developed methods which virtually eliminate the slice-to-slice in- consistency disrupting current KZM reconstruction and degrading image quality. Our central hypothesis is that this KZM approach shown in feasibility studies can now be markedly improved technically to simultane- ously provide improved through-plane resolution and allow reduced scan time, resulting in an efficient tech- nique enabling improved performance of clinical prostate T2SE and DWI MRI. We propose to: 1. Develop techniques for rapid, high quality, multislice acquisition suitable for SR prostate T2SE. We will incorporate non-traditional RF excitation pulses for improved fidelity in kZ. We will use simultaneous multi- slice (SMS) to reduce scan time and statistical reconstruction to retain SNR. We will develop means to measure the small, 1-2 mm A/P prostate motion during the scan, allowing accurate motion correction. 2. Extend the SR methodology developed for T2SE to DWI of the prostate. Segmented acquisition will provide for slice-to-slice positional consistency. SR reconstruction will be tuned to the individual b-values used. Sta- tistical reconstruction will retain SNR, allowing thin-slice images of the ADC. 3. Apply SR techniques to multislice prostate MRI. We will evaluate our technical developments in clinical evaluations, comparing conventional T2SE with our new SR method for a Super Abbreviated Exam (SAE). We will also evaluate our T2SE KZM method on improved accuracy in detecting PCa in the transition zone.

NIH Research Projects · FY 2025 · 2022-07

Abstract Obesity triggers cellular damage and impedes tissue recovery from injury, and its escalating prevalence may promote complications of peripheral vascular disease, such as critical limb ischemia (CLI) or renal artery stenosis (RAS). Reducing complications of obesity could diminish the risk of death, improve quality of life, and produce extensive cost savings. This application is based on the scientific premise that obesity increases tissue susceptibility to injury by interfering with normal defense and repair processes associated with mesenchymal stem/stromal cells (MSCs). MSCs constitute an effective endogenous cellular repair system, but obesity may blunt their efficacy. We found that obesity-induced MSC dysfunction in pigs was associated with altered mitochondrial structure and function, but the mechanisms of mitochondrial damage in human MSC and its contribution to regulation of MSC function in human obesity remain unknown. Our central hypothesis is that human obesity engages epigenetic mechanisms that impair human MSC mitochondrial structure and function and render MSC functionally deficient. We speculate that obesity alters in MSC the epigenetic states of micro-RNA (miR) miR-181a, a key miR that targets mitochondrial DNA and negatively regulates their function. A consequent fall in levels of the mitochondrial derived peptide (MDP) MOTS-c in turn impairs function and tissue repair capacity of MSC in obesity. To test our hypothesis, we will define gene expression and epigenetic states of mitochondrial targeting miRNAs and MOTS-c in human adipose tissue-derived MSC and elucidate their functional significance for both MSCs and their mitochondria. Our Specific Aims will pursue 3 hypotheses. Aim 1: Human obesity induces MSC miR-181a expression and in turn mitochondrial and MSC structural damage and dysfunction. Using RNA-seq we will identify miR-181a as a key miR upregulated in MSCs from patients with obesity vs. healthy controls. Its role in regulating MSC and mitochondrial function and structure will be assessed in vitro and in vivo (in mice with CLI or RAS) using novel in vivo imaging and ex vivo techniques. Aim 2: Human obesity engages epigenetic mechanisms to alter miR- 181a. We will define the epigenetic landscape of miR-181a using MeDIP-seq, and its contribution to MSC repair in vitro and in vivo using an epigenetic modifier. Aim 3: A fall in MOTS-c owing to mitochondrial damage contributes to functional impairment of ‘obese MSC’. Using novel MDP-seq we will pinpoint MOTS-c as a unique MDP linking mitochondrial to cellular integrity in MSC. MSC treated with MOTS-c peptide or neutralizing antibody will be characterized, and restoration of ‘obese’ MSC function tested both in vitro and in vivo. The proposed studies, employing cutting edge techniques, may uncover novel mechanisms underlying cell damage and impaired repair in human obesity. These studies will advance understanding of the pathogenesis of cellular damage, and likely contribute towards management of patients with obesity and vascular disease.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT The Mayo Clinic Immunology Ph.D. Training Program seeks to train leaders for the next generation of immunologists. This training grant proposal requests support for 6 pre-doctoral students who will be trained in a productive biomedical research program supported by a premier academic not-for-profit medical institution. The highly productive training faculty provide a rich training environment with basic and clinical investigators combining studies of fundamental immunology with translational potential. The training program has an exemplary track record in preparing students to succeed in scientific careers. Of all students who have successfully completed our Ph.D. Training Program , 98.9% have remained in science, science-related fields or medicine. 54.9% of those graduates currently hold academic rank, a rate almost twice the national average. Students in the program receive advanced training in primary literature and cutting edge technology focused coursework and electives in a multidisciplinary setting. Milestones in the program place emphasis on each trainee mastering the skills needed to formulate critical questions, and devising experimental strategies to provide definitive insights into the questions being addressed. Students learn to communicate ideas and research findings effectively using oral and written formats and practice these skills in classroom settings, in grantsmanship courses, at national and international scientific meetings, and by publishing original research articles in scientific journals. The typical training period for trainees supported by this grant is 5.2 years, capped by mentoring for the next phases of their developing careers.

NIH Research Projects · FY 2025 · 2022-07

We are submitting this application in the program area of Pharmacological Sciences (PS). The primary goal of the PhD Training Program in Molecular Pharmacology and Experimental Therapeutics (MPET) in the Mayo Clinic Graduate School of Biomedical Sciences (MCGSBS) is the development of independent investigators capable of directing outstanding research programs in academia, industry, or other settings. The faculty is comprised of 28 well-funded, independent investigators who are drawn from departments and divisions across the institution and who focus on a continuum of research areas encompassing studies from basic molecular and genetic aspects of disease through drug discovery and development of novel therapies for diseases. The faculty provides training opportunities in areas that include systems pharmacology, artificial intelligence, computational chemistry, molecular mechanisms of drug action and resistance, metabolomics, novel therapeutic strategies, the genetics of addiction, preclinical and clinical pharmacology, and pharmacogenomics of genes associated with drug responses. Forty-six predoctoral students (PhD and MD/PhD in PhD training), from varied scientific backgrounds in pharmacology, are currently enrolled in the MPET PhD training program. A rigorous didactic curriculum includes a series of Core Curriculum courses that ensure strong fundamental knowledge in biochemistry, molecular biology, genetics, statistics, cell biology and pharmacology, as well as a series of tutorial-based courses that provide advanced training in pharmacological sciences. During their first two years of study, students complete laboratory rotations and select a laboratory for their thesis research. They complete comprehensive written and oral qualifying examinations by the end of Year 3. Training in rigor and reproducibility is woven throughout the training program, and students are expected to meet the highest standards of scientific integrity. After developing a written thesis proposal delineating the questions and approaches to be pursued in the thesis research, the Thesis Advisory Committee reviews the proposed research at the first committee meeting. Students present work-in-progress updates on their research projects to MPET faculty and students each year. Trainees are required to develop independence and to publish at least one first-author article. In the last 15 years, MPET trainees have averaged over 2.2 first author articles and 5.6 total articles. Starting in Year 3 and beyond, students meet with their Thesis Advisory Committees at least twice per year. The average time to completion of the PhD is 5.9 years. Graduates of the MPET PhD training program have outstanding track records, with many students establishing new research directions that have long-lasting impacts on the mentor’s research programs. They go on to postdoctoral fellowships, with many now serving as principal investigators in academia and industry. We request 6 T32-funded positions.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Moderate (<60%) O2 (hyperoxia) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via effects on airway smooth muscle (ASM), a cell type that also contributes to impaired bronchodilation, and remodeling (proliferation, altered extracellular matrix (ECM)). Thus understanding mechanisms by which O2 affects bronchial airways is critical for therapeutic strategies in a vulnerable population. We focus on a novel, targetable mechanism in ASM: cellular senescence (Sen). Sen cells are long-living, and secrete factors (senescence-associated secretory phenotype; SASP) that promote inflammation and fibrosis via paracrine effects on naïve cells. Appeal lies in novel drugs that kill Sen cells (senolytics) such as dasatanib+quercetin (D+Q) and fisetin. Little is known regarding Sen cells in perinatal airways but our data indicate moderate O2 enhances detrimental Sen in human fetal ASM (fASM) with increased inflammatory, pro- fibrotic SASP that promotes proliferation and ECM of naïve ASM: effects inhibited by D+Q. We find that ROS and ER stress promote fASM Sen, and in newborn mice exposed to O2 (which results in AHR and fibrosis) D+Q alleviates O2 effects. Thus, we hypothesize perinatal O2 induces detrimental Sen cell burden that, via SASP, initiates and promotes AHR and remodeling: effects alleviated by senolytics. We propose 3 Aims using human fetal lung and in vivo neonatal mouse models of O2: Aim 1: Determine mechanisms by which hyperoxia induces cellular Sen in developing human ASM; Aim 2: Determine the role of cellular Sen in hyperoxia effects on developing human ASM; Aim 3: Determine effects of detrimental Sen on contractility and remodeling in mouse model of neonatal hyperoxia. In Aims 1 and 2, we will use 18-22 wk gestation human fASM and lung slices to examine mechanisms of Sen induction, focusing on ROS, mitochondria and ER stress (Aim 1) and downstream effects of Sen/SASP in the context of contractility and remodeling (Aim 2) following 40% O2. Alleviation by senolytics D+Q or fisetin (Aim 1, 2) are explored. In vitro studies are integrated in the newborn mouse model (Aim 3) where extent of Sen is assessed, and alleviation of airway hyperreactivity and remodeling by senolytics are tested. Clinical significance lies in establishing detrimental Sen in O2 effects on developing airway towards future therapeutic targeting for neonatal asthma.

NIH Research Projects · FY 2025 · 2022-06

Disrupted sleep, a major public health issue, independently increases risk for cardiovascular disease (CVD). Blacks have increased rates of sleep deficiency, which are likely under-reported. Importantly, these data relate primarily to those of West African ancestry. Our current knowledge of sleep disruption in Blacks, already severely limited in scope, cannot be readily applied to Somali Americans. The majority of Somali immigrants have settled in Minnesota, placing our Minnesota-based research team in a unique position to comprehensively study the mechanisms and consequences of disrupted sleep as a mediator of cardiovascular health disparities in this population. Our preliminary data suggest that Somali Americans have a high likelihood of disrupted sleep, which may put them at increased risk for hypertension and other CVD. Underlying sociocultural, behavioral, environmental and biological factors likely contribute to an increased risk for sleep deficiencies. We therefore propose an inter-disciplinary approach using a socioecological model informed by the National Institute on Minority Health and Health Disparities (NIMHD) Research Framework to determine the types and severity of undiagnosed sleep deficiencies in otherwise healthy Somali Americans, identify mechanisms contributing to their disrupted sleep, and examine the role of sleep deficiencies in raising blood pressure (BP). Our central hypothesis is that Somali Americans will have a high likelihood of sleep deficiencies attributable in part to unique multilevel individual, psychosocial, contextual and behavioral factors, which exert deleterious biological effects. We propose the following aims: Aim 1: Determine the types and severity of previously undiagnosed sleep deficiencies in otherwise healthy Somali Americans. Hypothesis 1: Somali Americans have a high (>50%) likelihood of previously undiagnosed sleep deficiencies (short sleep (<6 hours), insomnia and obstructive sleep apnea). Aim 2: Apply the NIMHD Research Framework to define psychosocial, behavioral, environmental and biological mechanisms mediating sleep deficiencies in Somali Americans. Hypothesis 2: Unique multilevel individual, cultural and environmental risk and protective factors play a mechanistic role in mediating an increased likelihood of disrupted sleep in Somali Americans. Aim 3: Examine the relationship between sleep deficiencies and increased BP in Somali Americans. Hypothesis 3: BP during wakefulness and/or sleep will be increased in those subjects with disrupted sleep, commensurate with type and severity of sleep deficiency, and moderated by factors such as sex and age. The expected outcome of this proposal will be a mechanistic pathway incorporating the NIMHD Research Framework to identify psychosocial, behavioral, contextual and biological factors mediating sleep deficiencies and related increases in BP, and consequently hypertension risk, thus addressing important knowledge gaps in understanding sleep-related health disparities and their consequences in Somali Americans.

NIH Research Projects · FY 2026 · 2022-06

Project Summary Cellular senescence is a programmed growth arrest activated by irreparable extrinsic or intrinsic stresses. Senescence can be beneficial in certain circumstances, such as tissue homeostasis during embryonic development or tumor suppression. However, if persistently secreted by senescent cells, the proinflammatory cytokines, chemokines, proteases, and growth factors are actually major drivers for aging and age-associated diseases and, paradoxically, promote tumorigenesis. Genetic or pharmacological clearance of senescent cells effectively improves lifespan and healthspan in rodent models. As such, targeting senescence has emerged as a promising therapeutic strategy to prevent or treat aging comorbidities and cancer. However, how the irreversible senescence program is induced and maintained in stressed cells remains poorly understood. Cells utilize primary cilia to convert environmental cues into diverse cellular signalings that govern proliferation, differentiation, and tissue homeostasis. Cilia dysfunction leads to a wide spectrum of syndromic disorders that are collectively termed ciliopathies. Using irradiation, we discovered that stressed human fibroblasts or epithelial cells exhibit transient cilia biogenesis. Strikingly, FBF1, a component of transition fibers (TFs) at the ciliary base, unexpectedly translocates to promyelocytic leukaemia nuclear bodies (PML-NBs) in stressed cells. PML-NBs are highly dynamic proteinaceous nuclear structures with instrumental roles in regulating stress-induced responses, including senescence and apoptosis. FBF1 depletion effectively abolishes stress-induced PML-NB upregulation and associated senescence initiation, whereas FBF1 overexpression shows the opposite effects. Our initial studies indicated that the stress-induced PML-NB translocation of FBF1 is regulated by a distinct cilia module comprising Joubert syndrome proteins ARL3 and ARL13B and the SUMO-conjugating enzyme UBC9. Further proteomic studies revealed novel FBF1 interactors (PML, 53BP1, and BRD4) implicated in PML-NB biogenesis and/or function. Remarkably, Fbf1tm1a/tm1a mice exhibit a significantly reduced senescence burden throughout life and could be further protected against irradiation-induced frailty. Our preliminary data thus suggest an exciting paradigm that a stress-induced TF-to-PML-NB translocation of ciliary protein FBF1 is essential for senescence initiation in mammalian cells. Here, we propose to use complementary approaches to address mechanistic questions, including how the ciliary ARL3-ARL13B-UBC9 module regulates FBF1 SUMOylation and PML-NB translocation (Aim 1), and how PML-NB-associated FBF1 promotes senescence in stressed cells (Aim 2). Together with the extended analysis of the physiological importance of FBF1 pathway in in vivo senescence mouse models (Aim 3), this project will potentially bridge the fundamental discovery to the next generation of therapeutic strategies for preventing or treating senescence-associated pathologies.

NIH Research Projects · FY 2026 · 2022-06

Cellular senescence is now recognized as one of the fundamental aging mechanisms contributing to multiple age-related degenerative conditions, including osteoporosis. In previous studies, we have systematically identified senescent cells in the bone microenvironment and demonstrated a causal role for senescent cells in mediating age-related bone loss in mice. In recent studies, we used a novel transgenic mouse model, p16- LOX-ATTAC, capable of temporal- and cell-specific senescent cell clearance, and found that in contrast to global clearance of senescent cells using the (p16)-INK-ATTAC model, clearance specifically of senescent osteocytes only partially replicated the beneficial skeletal effects of global senescent cell clearance, suggesting an important role for other cells in the bone microenvironment (e.g., immune cells) in contributing to skeletal aging. In addition, in our previous work, we demonstrated a dramatic upregulation of the senescence- associated secretory phenotype (SASP) in bone marrow myeloid cells with aging, and more recent studies by our investigative team have shown that with aging, activated neutrophils can induce senescence in multiple tissues in a paracrine manner. Conversely, senescent cells are capable of attracting neutrophils, which then further propagate senescence to other cells. Collectively, these studies point to previously unexplored cross- talk between skeletal and immune cells, specifically in the context of cellular senescence. Thus, our central hypothesis is that senescence of immune cells contributes to skeletal deterioration and conversely, senescent skeletal cells attract and contribute to an inflammatory and/or senescent phenotype of immune cells. We will test this hypothesis by examining the effects of senescent immune cells on bone and in the reverse experiment, evaluating the effects of senescent skeletal cells on immune cells. Our proposed studies make use of novel mouse models: p16-LOX-ATTAC mice, developed in the Khosla/Monroe laboratory, which are capable of temporal- and cell-specific (when crossed with a Cre mouse) senescent cell clearance; and Ercc1-/fl mice, developed by Drs. Niedernhofer and Robbins (Co-Is), where we can induce a tissue-specific DNA repair defect leading to premature cellular senescence only in that tissue (e.g., immune or skeletal cells). Collectively, our studies will address a number of fundamental questions relevant to osteoimmunology: (1) What are the specific populations of bone marrow immune cells that undergo senescence with aging using strictly defined criteria for cellular senescence (rather than the much broader umbrella of “immunosenescence” that includes inflammatory, but not necessarily senescent cells); (2) Does chronological or premature aging of the immune system cause skeletal deterioration?; (3) Conversely, do senescent skeletal cells lead to senescence, or at least inflammation, in bone marrow immune cells and does this further propagate senescence to other skeletal cells and perhaps systemically?; and (4) What are the potential mediators of the cross-talk between senescent skeletal cells and senescent/inflammatory immune cells?