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Project Summary After further deliberation from the ‘Targeted Challenge’ Working Group at NIH. It was determined that additional animals are required for the Programmable Delivery System effort, to be generated by three breeding cohorts of sows. Therefore, this administrative supplement reflects the same project summary, narrative, and research strategy as 3U42OD035738-03W1. The objective of this supplement project is to perform a side-by-side assessment of various gene editing (GE) formulations to identify those most effective as reagents for tissue editing. The project involves the delivery of CRISPR/Cas9 gene editing ribonucleoprotein complexes intravenously to pigs. Fluorescent reporter pigs will be used to detect editing activity and cell transduction efficiency. Successful targeting in cells and tissues by the formulations will be demonstrated by the induced expression of a red fluorescent protein (tdTomato). The effort for this project is structured into three parts: 1) In Vitro evaluations of test reagents, 2) In Vivo Toxicity Pilot delivery, and 3) final In Vivo Delivery to complete the animal studies. Importantly, all the reagents will utilize the same guide RNA from a common source that will target the same sites for editing. After each delivery, the pigs will be monitored daily, and blood will be drawn frequently. Inflammatory cytokines will be measured as well as serum chemistry levels and blood CBC and differentials, using the standard toxicology package provided by the University of Missouri Veterinary Medicine Diagnostic Laboratory (VMDL). At the close of the study (4 weeks ±2 days post-delivery), animals will be euthanized, and gross necropsies will be performed by the Testing Center staff. Tissues will be harvested, fixed, embedded, and sectioned for histopathology evaluation and imaging of tdTomato (or other immunostaining, based on project needs). For ‘Programmable Delivery’, three project-defined target tissues, along with the Liver and Thoracic Dorsal Root Ganglion will be evaluated. Ultimately, these evaluations will include H&E, cell-specific markers in serial sections to determine which cell type(s) were transduced, and high-resolution fluorescent imaging of the most targeted tissues. A detailed summary of the imaging assessments, blood panels, and circulating inflammatory markers will be provided to the targeted challenge board for their rankings. At the end of the study, the results and tissues will be provided to the submitting investigator teams.

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PROJECT SUMMARY The goal of the “Nutrition for Precision Health (NPH), powered by All of Us” consortium is to generate a rich database from a representative population to develop a first-of-its-kind diet prediction algorithm. The aim of the Louisiana Nutrition for Precision Health Center (LA-NPH), consisting of LSU-Pennington Biomedical (PBRC) in Baton Rouge and LSU-Health Sciences Center (LSUHSC) in New Orleans, is to participate in the NPH consortium as a clinical center to recruit, enroll and retain participants from All of Us in the three planned study modules. In Module 1, 900 participants from the All of Us Research Program in Louisiana (Baton Rouge and New Orleans) will be enrolled in a 10-day prospective, observational study. Following the completion of Module 1, 145 study participants who meet specified eligibility criteria for enrollment to the controlled feeding studies, will participate in Module 2 and 60 in Module 3 (PBRC only). Modules 2 and 3 are randomized cross-over nutritional intervention studies to evaluate the individual response to three, 14-day isocaloric diet interventions. Module 2 is conducted in community dwelling participants while Module 3 is conducted in domiciled participants. The goal of this administrative supplement is to support enhanced efforts of LA-NPH in increasing enrollment targets in all 3 modules with 960 in Module 1, 205 in Module 2 and 65 in Module 3.

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The NIH-funded Nutrition for Precision Health Study (NPH) is a discovery science project established in January 2022 for the purpose of elucidating variability in individual response to dietary exposures. The study is the first ancillary study of the All of Us Research Program (AoURP). The University of North Carolina at Chapel Hill is home to one of the six clinical centers in the NPH consortium (UNC-CC, NIH-UG1HD107692, MPIs Mayer-Davis and Tate). NPH participants, all of whom are participants in AoURP, begin with NPH Module 1 which is a cross-sectional study designed to carefully characterize dietary intake, a wide array of behavioral and social factors, as well as physiological measures including assessments during a liquid mixed meal tolerance test. Module 1 assessments occur over an 8-10 day period. A subset of participants who have completed Module 1 then go on to participate in either Module 2 or Module 3, both of which involve a controlled feeding study to assess response to each of three different test diets, using a cross-over design. Each diet is consumed for a period of two weeks, with a minimum of a two week washout period. At the UNC-CC, we offer only Modules 1 and 2 (free-living); not Module 3 (domiciled). Our clinical center is co-located in Chapel Hill and in Kannapolis, NC, approximately two hours apart by car. We are unique in that we established an AoURP recruitment center for the purpose of conducting the NPH study. As we have been successful both in enrolling participants into AoURP and then recruiting participants into Modules 1 and 2, the UNC-CC proposes to build upon our primary infrastructure to increase our sample size and thereby contribute to the national consortium as we seek to ensure the overall success of the study. Specifically, we request additional funds to increase enrollment as follows: • Module 1: 1145 participants (increase of 145 participants from our initial enrollment target) • Module 2: 430 participants (increase of 20 participants from our most recent enrollment target) We propose an enrollment split with approximately 50% of participants seen in Chapel Hill and 50% of participants seen at the Nutrition Research Institute (NRI) in Kannapolis with most of the increased enrollment target for M2 to be enrolled at NRI. The NRI is well suited for this expansion given the capacity of the metabolic kitchen at NRI to accommodate the majority of these additional M2 participants.

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PROJECT SUMMARY Epidemiological and clinical studies support an important role of nutrition in health. However, nutrition research is limited by bias due to self-reported diet data and inter- and intra-individual variation. High-throughput `omic' profiling techniques combined with advanced remote real-time data collection now enable comprehensive studies of individual responses to diet thereby creating opportunities for personalized nutrition advice. Our overall goal is to facilitate the Nutrition for Precision Health (NPH) Consortium by leveraging our existing Illinois Precision Medicine Consortium (IPMC) infrastructure to enroll All of Us Research Program (AoURP) participants in the discovery nutrition science study involving three diet modules. Proposed is investigation of specific elements of a Dietary Approaches to Stop Hypertension (DASH) diet with blood pressure (BP) as the primary outcome. BP is regulated by a complex network of mechanisms under the influence of genetic and environmental factors, and high BP is recognized as the leading modifiable risk factor for cardiovascular disease, cerebrovascular disease, kidney disease and all-cause mortality worldwide. DASH diet adherence has consistently documented reduced BP, independent of baseline calorie or sodium intake. Although older, hypertensive and Black persons show greater BP responses to DASH adherence and reduced dietary sodium intake, inter-individual variability is observed and remains unexplained. In Module 1, we will follow 2,000 AoURP participants for 14 days to examine baseline diet and physiological responses to test-meal challenges hypothesized to elicit variable physiological and metabolomic responses based on individual cardiometabolic, genetic and gut microbial status. We then examine responses to three 14-day intervention periods involving DASH-type diets among 400 consenting Module 1 participants in a free-living controlled feeding study (Module 2) and in 200 Module 1 participants in a domicile controlled feeding study (Module 3). The three intervention diets are isocaloric, sodium equivalent and include: i) DASH-Standard, ii) DASH-FFF, specifying fruits, flavonoids and fat and DASH-PP, emphasizing plant protein. Each diet has distinctive nutritive properties that influence BP regulation and each will elucidate diverse physiological, metabolomic, and microbiomic responses that are modified by cardiometabolic and genetic status. Our three IPMC clinical sites including Northwestern University, the University of Chicago, and the Illinois Institute of Technology span wide yet geographically distinct service areas in ethnically and socioeconomically diverse Chicago communities, thereby offering targeted enrollment of demographically diverse participants. This research in collaboration with the NPH consortium initiates fundamental causal and mechanistic insight into the role of the DASH-type diet in BP regulation with potential to discover novel biological pathways underlying risks for developing high BP. This advanced knowledge can inform unprecedented personalized diet recommendations to prevent and treat the massive public health burden off hypertension.

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ABSTRACT The reasons for individual variability in the physiologic response to dietary patterns are not well understood but hamper efforts to provide optimum diets to our population. There is an urgent need to understand the complex interaction of demographic, genetic, metabolic, behavioral, psychosocial, and environmental factors that affect the responses to dietary patterns in order to prevent and treat nutrition-related chronic diseases. The field of “precision nutrition” holds great promise for elucidating these interactions to eventually predict the optimal diet for an individual or groups of individuals. The overall objective of this application is to request additional funds for the final year of subject recruitment at the University of Alabama at Birmingham (UAB) to support meeting enrollment targets for the Nutrition for Precision Health Consortium. Due to delays in protocol development and a slow enrollment rate at the beginning of the study, additional staff effort is needed in Year 5 to increase enrollment to meet our targets. The study team will collect a wide range of physiological and metabolic data from individuals in response to free-living (module 1) and controlled diets (module 2), that will be used in analyses to determine potential predictors of response to diet. Sophisticated data methods (artificial intelligence, machine learning, mathematical modelling) will then be employed by the study group to identify the comprehensive phenotypes needed for individualizing diet prescriptions. We aim to accomplish the following two specific aims: Specific Aim 1 (module 1): Conduct an observational study of 1000 free-living individuals consuming their usual diet for 8-10 days. The physiologic responses to a standardized test meal challenge will be assessed while they are consuming their usual diet. Specific Aim 2 (module 2): Conduct a free-living controlled feeding study in 250 subjects fed three isocaloric diets varying in macronutrient composition at maintenance energy requirements. Diets are designed to elicit a wide range of responses among participants. The physiologic responses to standardized test meals and diet-specific meals will be measured at the end of each 14-day diet period. We will also collect measures of 24-hr glucose, 24-hr physical activity and sleep during each diet period. UAB, with access to >16,000 All of Us participants in Birmingham, outstanding facilities for conducting diet interventions, and an outstanding research team, can support the Nutrition for Precision Health Consortium with meeting enrollment targets with additional funds.

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PROJECT SUMMARY The All of Us New England Clinical Center (AoU-NE-CC) is a collaboration of New England–based research teams with the necessary expertise and resources in nutrition and clinical translational science to implement successfully all 3 modules of the NIH Common Fund’s Nutrition for Precision Health (NPH) Clinical Center program. The Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (Tufts- HNRCA) and Massachusetts General Hospital (MGH) will lead the AoU-NE-CC and partner with All of Us New England to ensure the diversity of participants and scientific rigor required to identify inter-individual variability of response to dietary patterns. The AoU-NE-CC is committed to All of Us core values and has implemented best practices of team science throughout its development. The AoU-NE-CC will serve as a key partner in the development of a rigorous NPH common protocol and examine habitual dietary intake (Module 1), measure physiological responses to a mixed-meal challenge (Modules 1-3), and identify responses to 3 intervention diets in both free-living (Module 2) and domiciled (Module 3) controlled feeding conditions. Coupled with standard All of Us data, the physiological responses collected through these modules will be used to develop predictive algorithms that inform precision nutrition approaches for long-term health. To attain maximum metabolic and microbiome response variability, we propose the following 14-day isocaloric diets for Modules 2 and 3, separated by 4-week washout periods: (1) a high-adherence Dietary Approaches to Stop Hypertension (DASH) diet; (2) a low-adherence DASH diet; and (3) a ketogenic diet. We also propose the use of the thoroughly tested mixed-meal challenge PhenFlex, which can be standardized across clinical centers to provide a multisystem assessment of metabolic flexibility. With its outstanding core facilities, including metabolic kitchens and domiciled feeding and nursing centers, as well as a rich legacy of conducting rigorous feeding studies, the AoU-NE-CC is uniquely skilled and positioned to serve as a clinical center for the NPH. Additional strengths of the AoU-NE-CC include extensive experience in recruiting clinical populations and biobanking, engaging volunteers through community outreach, active participation in global precision nutrition initiatives, and an outstanding track record of productive multisite collaborations in nutrition, omics, and precision health. Importantly, the Tufts-HNRCA and MGH teams are closely aligned with the All of Us New England team and its record of high participant recruitment and retention and its diverse cohort, which will support the NPH consortium goals. By partnering with NPH and All of Us in this novel modular discovery science study, the AoU-NE-CC and its experienced, forward-thinking, highly collaborative team will contribute to the development of precision nutrition approaches that support optimal health across the adult lifespan.

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Contact PD/PI: GANTZ, Marie G. PROJECT SUMMARY/ABSTRACT From Proposal: The overarching goal of the RTI International–Cornell Research Coordinating Center (RCC) is to provide seamless operational support and multidisciplinary experience for building consensus in the Nutrition for Precision Health Consortium (NPHC). In the 1-year planning phase, we will prioritize efficiency and objectivity in facilitating the design of diet modules (1: usual dietary assessment; 2: controlled feeding dietary intervention; and 3: domiciled dietary intervention) nested in All of Us and their research protocols. In the 4-year implementation phase, we will facilitate the implementation of Modules 1–3, enable the flow of quality data and specimens across the NPHC, and integrate curated, Artificial intelligence (AI)–ready data into the All of Us Researcher Workbench, using our established data coordination processes and systems. Innovations in our approach include tools for conducting dietary studies in hard-to-reach populations, idiographic (or subject-as-own-control) clinical trials, and wearables research tools and analytics. Specifically, the proposed RCC will excel in administrating and coordinating NPHC and its research initiatives, clinical interventions, and data and biospecimen sharing, as follows: Aim 1: Optimize the scientific rigor of Modules 1–3 Aim 2: Ensure NPHC study data are FAIR (Findable, Accessible, Interoperable, and Reusable) Aim 3: Enable seamless data collection, curation, and transfer including to the Researcher Workbench Aim 4: Minimize time to launch of Modules 1–3 Aim 5: Maximize the efficiency of consortium communications and collaborations NPHC is being launched to address major research gaps in nutrition; the nature of these gaps and the complexity of the NPHC activities cannot be underscored enough. RCC will be led by Multiple Principal Investigators with complementary expertise in coordinating center and multisite leadership and biostatistics (Dr. Gantz at RTI) and nutritional intervention and clinical expertise (Dr. Mehta at Cornell University) with the support of a team organized around cores for Design and Analytics, Data Curation and Systems, and Study Implementation. Other key components include single IRB and medical safety monitoring. The RCC will also benefit from our collective institutional strengths, with an expert pool of wide-ranging research and clinical backgrounds pertinent to the NPHC (e.g., omics, bioinformatics, AI, clinical trial intervention, nutritional assessment). The proposed RCC is immediately and amply prepared to support to NPHC in its mission to develop clinically meaningful algorithms that predict individual responses to food and dietary patterns and to improve health across the U.S. population. Project Abstract

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Abstract Breast cancer is the most commonly diagnosed cancer among women worldwide. Patients diagnosed with breast cancer face the important question of what therapeutic regimens to choose, and they are eager to know the effects of potentially applied treatments. While treatment decisions have become more refined over time, they are not personalized and as such patients may still be over or undertreated. There is a paramount need to integrate the multi-scale, multi-modal, and multi-timepoint patient data and to build the capacity of systematically and accurately assessing a patient’s individual data to guide precision treatments of breast cancer. The complexity of multi-modal datasets poses challenges for physicians to interpret and integrate information, where artificial intelligence (AI) and data science are capable of extracting, aggregating, and inferring predictive insights. The goal of this study is to develop ethically designed AI prediction models using multi-modal data (clinical variables, medical images, and genomics assays, from individual, macro-scale, to micro-scale and longitudinal) to assess treatment efficacy of breast cancer and guide precision treatment decision-making. We propose establishing a multi-center collaboration network (University of Pittsburgh, Duke University, and MD Anderson Cancer Center) to curate diverse patient data for the AI model development and evaluation. We have assembled an experienced multi-disciplinary team with data scientists, oncologists, radiologists, geneticists, surgeons, pathologists, biologists, and biostatisticians. We propose to study two specific aims to demonstrate the concept by year 2 through establishing the team and collaboration network, delivering AI models, contributing new AI techniques, and crafting plans for continued work.

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Precision medicine aims to accurately classify patients to improve diagnosis, intervention selection, and prognosis. The All of Us Research Program (AoURP) collects an array of data types from participants, including surveys, electronic health records (EHRs), physical measurements, wearable devices, and biosamples, offering valuable insights into health trajectories. However, certain aspects of a participant’s life remain missing in the collected data, which can limit the accuracy of research and care. To address this gap, we propose the creation of the All of Us Center for Linkage and Acquisition of Data (CLAD) to supplement existing data sources using passive data streams and deploy integration strategies to "put the patient back together again" and more deeply assess health outcomes. This team brings together collective experience leading large initiatives involving data acquisition, linkage, harmonization, quality assurance, pipelines and platforms, governance, and security. We will design and implement a data collection, linkage, and integration strategy that lays a foundation for a variety of AoURP data linkages for identified, and de-identified data integration, including person-level linkages such as with mortality, residential history, and administrative claims, and geocoded data pipelines to enable linkages with environmental and economic data. The CLAD will acquire and process new data linkages and geocoded data in a cloud-based Data Linkage Platform (DLP), guided by our experience formulating researcher-ready datasets with scientific utility. Our CLAD team will perform data quality assurance, repair, and standardization checks to ensure accuracy and robustness of data-driven research. This endeavor will align data with interoperability standards and clinical terminologies, extend them where necessary, and create a data quality dashboard for every data change and data health check. We will also explore new methods of clinical data acquisition to mitigate data missingness by comparing data provided from recruitment sites with EHR data from Health Information Networks. CLAD data sources and novel analytical methods, such as probabilistic models, will be used to reveal patterns of care, health outcomes, and potential interventions for common, chronic, and genetic diseases.

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PROJECT SUMMARY This application requests funds to purchase a cellenONE X1 Neo single-cell isolation and liquid dispenser system from Cellenion. The proposed instrument will be located in the Mass Spectrometry Proteomics Core at Baylor College of Medicine. The cellenONE X1 Neo is essential for establishing ultra-low-input proteomics capabilities at BCM and will be primarily used to isolate single cells and subcellular structures from clinical samples, pre-clinical patient-derived xenograft models, and various animal- and cell-based model specimens. Configured with protein sample processing workflows for bottom-up mass spectrometry, this platform will meet critical sample preparation needs for picogram- and nanogram-level starting materials, which demand extremely precise, low-volume, and contamination-free sample handling not adequately supported by our standard core protocols. Single-cell and spatial proteomics is a novel and rapidly advancing area of biomedical research with the potential to transform our understanding of cellular heterogeneity and disease mechanisms. However, despite BCM’s extensive research infrastructure, this capability is currently lacking at our institution. The addition of the cellenONE X1 Neo represents a significant leap forward, enabling our core to offer a complete, automated solution for ultra-sensitive proteomic analysis. Importantly, this instrument will leverage a recent acquisition of the Bruker timsTOF Ultra2 in the Mass Spectrometry Core – a $1.2 million investment in state-of-the-art mass spectrometry instrumentation capable of measuring ultra-small-scale proteomes. Although this Bruker timsTOF is already used for other challenging applications, the absence of a suitable single-cell preparation platform remains a critical barrier to adopting true single-cell and spatial proteomics workflows. The unifying aim of the projects supported by the cellenONE X1 Neo is to explore the molecular mechanisms driving normal physiology and disease at the level of individual cells or rare cell populations. This instrument will provide the precision, scalability, and operational robustness necessary to meet the evolving needs of our growing user base. Its integration into the Mass Spectrometry Proteomics Core aligns with BCM’s strategic plan to expand and share cutting-edge proteomics capabilities and will directly enhance research initiatives in cancer, metabolic disease, neuroscience, immunology, and beyond. The cellenONE X1 Neo will position the Core, and BCM more broadly, as a regional leader in ultra-sensitive proteomics by providing a comprehensive, accessible sample processing solution.

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PROJECT SUMMARY/ABSTRACT The Division of Translational Radiation Sciences (DTRS) was established to accelerate the discovery and clinical implementation of new therapeutic strategies in clinical radiotherapy at the University of Maryland School of Medicine. DTRS and the Department of Radiation Oncology have been at the forefront of this field for several decades, having been one of the first institutions to secure a Medical Countermeasures Against Radiological Threats (MCART) consortium award from the NIH in 2005. DTRS currently leads two NIH-sponsored consortia: the Intercollaborative Radiation Countermeasures (INTERACT) Consortium (5U19AI150574-05), and the Radiation Oncology-Biology Integration Network on Oligometastasis (ROBIN OligoMET, 5U54CA273956-03). Our experienced physicists perform both in vitro and in vivo irradiations (in small and large animals) for investigators involved in these two consortia, as well as for other researchers requiring precise and accurate delivery of radiation doses in their experiments. DTRS is not a core facility but operates on a fee-for-service basis to perform and support these procedures for investigators across campus. A large number of our users rely on our current XRAD-320 X-ray irradiator, which has become increasingly unreliable, as the manufacturer no longer services the power generator or offers preventive maintenance, making future repairs potentially impossible. Our department also utilizes cesium-137 irradiators that must be phased out to comply with the U.S. Congress–mandated National Defense Authorization Act (NDAA), which calls for the elimination of all cesium- based irradiators in the U.S. by December 31, 2027, to mitigate national security risks associated with high- activity radioactive sources. We are therefore proposing to replace our aging and unsupported irradiator technology with a modern X-RAD 320 equipped with OptiMAX imaging. This state-of-the-art instrument offers advanced imaging capabilities, enabling precise targeting of radiation delivery to biological tissues. It will support current studies with improved accuracy, allow for the development of new experimental designs, and reduce labor requirements for existing protocols. Additionally, this system is considered one of the most suitable replacements for Cs-137 gamma irradiators. We have identified a group of NIH-funded users within the University of Maryland who rely on the current system or who would benefit from expanded capacity beyond what our aging platform can reliably support. Many of the research projects described in this application are translational in nature and aim to accelerate the understanding of disease mechanisms and the development of novel therapeutic strategies. Our institution is committed to providing substantial support for the installation and long-term operation of this system. The management and operational infrastructure are already in place, led by an outstanding technical team dedicated to delivering high-quality service to all users.

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Abstract The Ohio State University Comprehensive Cancer Center (OSUCCC) Genomics Shared Resource (GSR) is requesting funds to purchase an Oxford Nanopore Technologies (ONT) PromethION 24 long-read nucleic acid (DNA and RNA) sequencer. There are several innovative and exciting features of the PromethION 24 which is the “third generation” sequencing instrument that best meets the needs of OSU researchers. Features include 1: the ability to assess native DNA and RNA modifications; 2: the ability to generate long-reads of a variety of lengths from a few kb to over 2 Mb of continuous sequence; 3: the ability to perform long-read single cell sequencing from 10X Genomics single-cell libraries enabling evaluation of expression of mRNA isoforms, variants and mutations from single-cells; 4: adaptive sampling which is on-instrument targeted sequencing strategy in which nucleic acids not of interest are rejected from the pores; 5: the ability to run up to 24 flow cells synchronously or asynchronously which adds flexibility in project management and ability to run multiple types of projects at the same time as well as being able to easily accommodate larger projects. The PromethION has advantages over long-read instruments from other companies because of it being able to sequence longer (>25 kb) of contiguous sequence and the ability to directly sequence RNA and interrogate multiple different RNA modifications. Furthermore, ONT offers a variety of on-instrument and cloud-based open-source software (free to users) that range from straight-forward “point and click” user-friendly tools to more sophisticated analytical programs. There are also links to community-based GIT-hub software on the ONT website. Seven major and six minor users have current NIH-funded projects that could benefit from having this technology on site. Examples of types of projects that would benefit from access to a PromethION 24 on site include: single-cell RNA- sequencing to identify allele-specific expression and escape from X-chromosome inactivation, single-cell isoform expression, long-read sequencing of single-cell spatial (10X Visium) B-cell receptors (BCR)/T-cell receptors (TCR), long-read telomere sequencing (Telo-Seq), long-read ribosomal RNA sequencing to understand ribosomal RNA processing, long-read DNA sequencing to understand genomic complexity at the Spinal Motor Neuron (SMN) locus and its impact on phenotype, and long-read direct RNA sequencing for transcript and isoform analysis in T-cells during parasitic infection. The Promethion 24 will be housed in the GSR with administration and financial support from the OSUCCC. The instrument will be available to all OSU investigators as well as outside investigators. Oversight of the instrument will be supported by the GSR technical director who has ONT experience as well as 3+ staff with NGS library expertise. There are currently no long-read PromethION 24 sequencers available in any shared resource at OSU so this instrument fills a critical need and will support cutting-edge applications and science.

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PROJECT SUMMARY/ABSTRACT: This S10 Shared Instrumentation Grant application from Washington University (WashU) in St. Louis requests funds in partial support of the purchase of a Sybmia Pro.specta X3 scanner (Siemens Medical Solutions USA). This hybrid single photon emission computed tomography and x-ray computed tomography (SPECT/CT) system will be housed in a dedicated Nuclear Medicine research facility for the non-invasive assessment of therapeutic and diagnostic (theranostic) radiopharmaceuticals. This state-of-the-art instrument will be a critical, broadly used resource for the clinical and translational neuroscience, cardiovascular and oncology research programmes at WashU. The requested SPECT/CT will be the only research dedicated SPECT/CT system across the WashU clinical enterprise. At present, WashU through its affiliated Hospitals, has access to 7 SPECT/CT scanners across the medical campus. These are dedicated for standard of care and clinical trial workflows, 1 of them being at the Children’s hospital (out-of-reach for research), and 2 of the SPECT scanners are obsolete and only used for planar imaging. These systems are all >10 yr, and they are heavily utilized, at nearly 8 h of scan time per day average (utilization >85%), which does not include protocol development and maintenance. Access for research is highly restricted and there is no support for the special attention required for clinical research. Additionally, in the greater St. Louis region beyond WashU there is no research SPECT/CT hardware, and the nearest research SPECT/CT scanners are located at University of Missouri Veterinary Health Center (2.5 h drive), dedicated for non-human use. The Symbia Pro.specta incorporates advanced workflows including advanced iterative data driven motion correction features are critical for advanced quantitative imaging-; a redesigned quantitative framework for therapeutic absorbed dose assessment; and best-in-the-field collimators. The requested SPECT/CT scanner will anchor major new research efforts in theranostics for cancer, cardiovascular disease and neuroscience at WashU. Towards this end, Pamela Woodard, Radiology Chair and MIR Director, and Timothy Eberlein, Director, Alvin J. Siteman Cancer Center, have made substantial financial and administrative commitments to ensure the successful utilization of this instrument. These include funds for (1) installation and renovation costs, (2) adjacent radioactive handling and patient-administration space, (3) maintenance for the instrument, (4) pilot funds for protocol development and (5) personnel support. A new Section of Medical Physics is being established to harness the outstanding imaging science and translational radiopharmaceutical expertise at WashU that will be co-located with this centerpiece scanner. The combination of advanced instrumentation and robust support from our institution will enable groundbreaking discoveries and innovations that will benefit both our research community and patients, reflecting our dedication to excellence in scientific inquiry and healthcare.

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PROJECT SUMMARY We propose to acquire an Avidion Cell Avidity analyzer from LUMICKS to support research on a broad range of biological questions at Yale University. It has long been understood that important physiological processes are regulated by the mechanical forces involved in cell interactions ranging from endothelial tight junctions to neuronal synapses to immune cell synapses. Yet, the ability to measure the strength of these interactions has been limited to highly technical, low throughput assays that probe only a single cell at a time. Less than a decade ago LUMICKS developed the z-Movi to measure cell-cell interaction strength on 100s of cells by acoustic force applied to a fluorescent cell interacting with a monolayer of cells on a glass plate. The PI, Samuel Katz, along with several other investigators at Yale have integrated this technology into our workflows and found it to provide meaningful measurements that accelerated our research. Based on this positive preliminary experience multiple other investigators have inquired about incorporating avidity measurements in their research programs, but the number of users that can complete an experiment on the current system each day is limiting. The recently developed LUMICKS Avidion has increased the throughput by an order of magnitude capable of approximately 200 measurements a day and two to three users. Moreover, it has expanded to four-color fluorescence in order to measure heterogenous cell populations and correlate Cell Avidity with other cell reporters. The automated nature of the Avidion with straightforward software for analysis further improves widespread usability. While this proposal nominally showcases 5 major users and 11 other users of this novel technology, multiple other investigators have expressed interest. The user base represents 12 different departments ranging from basic biologic inquiry to clinical translational uses. The requested instrument will be installed in a shared core facility at Yale, where it will support a large community of NIH-funded researchers studying cancer, autoimmunity, cardiovascular, neurodegenerative, inflammatory and other diseases. The absence of another commercially available or homemade alternative with the singular capabilities of the Avidion at Yale and the large NIH-funded userbase performing groundbreaking, high-impact research in a multitude of fields underscores the necessity for this advanced equipment.

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This proposal requests NIH funding for the procurement of a VisualSonics Vevo F2 LAZR X20 Multi-Modal Imaging system. The Vevo F2 LAZR X20 is a highly versatile imaging system specifically designed for non- invasive pre-clinical small animal research. This newer generation ultrasound/photoacoustic imager will replace the old generation Vevo 2100 LAZR system that has been phased out by Fujifilm VisualSonics. The new equipment will enable high resolution longitudinal investigation of anatomical and functional changes associated with disease progression and enable monitoring of therapeutic responses in a non-invasive manner. The instrument will support 8 major users, 5 minor users and 4 early-career investigators at the Massachusetts General Hospital’s Wellman Center for Photomedicine (Dermatology and Pathology), Neurosurgery, Cardiovascular Research Center (Medicine), and Athinoula A. Martinos Center for Biomedical Imaging. The diverse research applications of these researchers are focused on using imaging technology to understand the pathophysiology of an array of high impact diseases such as cancer, cardiovascular disease, abnormal brain function, neurological pathologies/injury, and antibiotic-resistant infections. Collectively, these scientists have experience using the most sophisticated optical imaging technologies currently available to biomedical research, each of which has its own intrinsic strengths and weaknesses. An internal advisory committee formed will ensure smooth operations, training, maintenance, compliance and resolve user time conflicts. A strong institutional and departmental support will ensure maintenance, support for core staff and other technical staff and a provision for training new research staff. There is no accessible photoacoustic system available to us where animals can be easily transported for any of the above applications. There are just two existing US/PAI systems in the Boston area – Vevo 3100 LAZR- X. However, the sales of this model has been discontinued by the vendor with official support ending in 2027, further limiting their ability to sustain or expand access for new users. This will be the first Vevo F2 LAZR X20 system in Boston. A few unique features of the new Vevo F2 LAZR X20 system are listed below: • An increased wavelength range (660-1320 nm) with increased laser power enabling photoacoustic imaging for treatment planning and monitoring at a depth not obtainable with other optical imaging technologies • Capability to customize configuration (waveforms and pulse sequences) for acoustic engineering • Real-time display of co-registered physiological and anatomical information of target tissues • Capability to perform 4D and whole-body imaging • Measure tissue oxygenation and hemodynamics • Monitoring physiological and anatomical changes in high resolution

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Vesicating (blister-forming) chemical-threat agents such as sulfur mustard (SM) or mustard gas, nitrogen mustard (NM), lewisite, and phosgene oxime can cause moderate to severe injuries and pain to the skin, eyes, and lungs. SM and NM are highly reactive bifunctional alkylating agents that can covalently modify all major cellular biomolecules, such as DNA, proteins, and lipids; thus, they are highly toxic. The eyes are particularly vulnerable to vesicant injuries, which cause a biphasic pathology of an acute response of photophobia, corneal erosions and inflammation, and chronic or late effects with significant deterioration of corneal structure and function from neovascularization, epithelial defects, fibrosis, and opacity. No therapeutic drugs are available as Medical Countermeasures (MCMs) for vesicant damage to the eye, eyelid or other organs. The major obstacle in developing potential MCMs is our limited understanding of the complex pathophysiological response of the eye after vesicant exposure. In this application, we propose to test the hypothesis that vesicating ocular injury pathology involves bioactive sphingolipid (SPL) pathways for acute and chronic inflammation and subsequent cornea, conjunctiva, and eyelid damage, causing significant vision impairment and dry-eye symptoms. In preliminary studies, we developed and characterized an NM-induced ocular surface injury (NMOSI) in mice, exposing the entire ocular surface to NM instead of only the cornea. We observed a severe acute inflammatory response that resolves in a month and cause damage to the cornea, atrophied eyelid glands, almost complete loss of vision, and apparent dry-eye symptoms. We found increased activity of acid sphingomyelinase, concurrent reduction in the sphingomyelin, and increased ceramides, suggesting sphingomyelinase activation in ocular surface tissue at three days post-exposure. Here, we propose to characterize NMOSI models in mice and rabbits, focusing on conjunctival goblet cells and epithelial stem cells and how NM affects the eyelids and their glands and causes dry-eye symptoms (SA #1). We will determine the temporal and spatial relationship of NM to SPL pathway for acute toxicity in ocular surface tissue of mice and rabbits separately from the cornea, conjunctiva-sclera, and eyelids at different time points (SA #2). It is unknown whether NM or SM-induced SPL pathways are overlapping. Hence, we propose to study if the NM- induced SPL pathway activation is similar to SM exposure (SA #3). Lastly, we plan to map out the pathway of SPL activation and lipid signaling using in vitro assays with meibomian gland epithelial and corneal cell lines (SA #4). We expect to identify novel associations of bioactive lipids in the inflammatory and wound-healing pathways of vesicating ocular injury, which will aid in improving our understanding of pathophysiological mechanisms of the injury and aid in developing potential MCMs in the future.

OD - NIH Office of the Director

PROJECT SUMMARY/ABSTRACT This shared instrumentation grant proposal seeks funding to upgrade the Bruker 7T preclinical MRI system at the UC Davis Center for Molecular and Genomic Imaging (CMGI), a vital resource supporting a broad community of NIH-funded investigators. The current Bruker AVANCE III console, installed in 2010, has reached end-of-life, with no guaranteed vendor support and limited spare parts, placing the system at significant risk for prolonged downtime. This vulnerability threatens a diverse portfolio of NIH-funded research at UC Davis, encompassing cancer biology, neuroscience, cardiometabolic disease, immunology, molecular imaging, translational nanomedicine, and multi-modal studies integrating MRI with PET and optical imaging. Upgrading to the fully supported AVANCE NEO console is essential to ensure continuity and reliability for these critical research programs. Seamless compatibility with the existing Bruker 7T magnet will enable a smooth transition, minimizing disruption to ongoing studies. With CMGI’s specialized infrastructure and technical expertise in in vivo preclinical imaging, and strong institutional backing for robust operation and long-term maintenance, this upgrade will not only safeguard current research but also empower UC Davis investigators to pursue innovative biomedical studies and accelerate the translation of preclinical discoveries into clinical advances.

OD - NIH Office of the Director

Project Summary We seek NIH support for the acquisition of an Asymmetric Flow Field-Flow Fractionation system coupled with Multi-Angle Light Scattering (AF4-MALS) to be housed in the Johnson Foundation Structural Biology and Biophysics Core (JFBSB Core) at the University of Pennsylvania. This platform will provide critical capabilities for the separation and label-free analysis of macromolecules and nanoparticles in solution, including lipid nanoparticles (LNPs), viral vectors (e.g., AAVs), protein-nucleic acid complexes, and phase- separated assemblies. The requested commercial instrument integrates two powerful technologies: 1. field- flow fractionation (AF4) for size-based, non-destructive separation of complex or fragile species and 2. multi- angle light scattering (MALS) for the direct measurement of molar mass, radius of gyration, hydrodynamic radius, and particle size distribution without reliance on calibration standards. Additional detectors, including differential refractive index (dRI), dynamic light scattering (QELS), and ultraviolet-visible absorbance, allow the system to rigorously quantify particle concentration, heterogeneity, and conjugation state in real time. These capabilities are essential for fully understanding the structure-function relationships of therapeutic macromolecular formulations and advancing gene delivery technologies. The proposed system will support the work of 10 NIH-funded projects in structural biology and nanomedicine. Projects will include structural optimization of LNP formulations for nucleic acid delivery, analysis of biologically relevant higher-order protein assemblies and aggregates, and separation of nucleoprotein complexes. This technology complements and enhances existing SEC-MALS, SAXS, and AUC platforms at Penn, enabling orthogonal workflows across the campus research landscape. No equivalent system currently exists at the University of Pennsylvania. The requested AF4-MALS system from Wyatt Technology offers unmatched integration with ASTRA software for advanced analysis, U.S.-based support, trade-in options for legacy systems, and the lowest risk of import-related tariff costs among evaluated vendors. The JFBSB Core, with a strong track record of S10 stewardship, will ensure broad access, expert support, and long-term sustainability. The requested instrumentation will have immediate and wide-ranging impact on federally funded research programs across Penn and its affiliated institutions.

OD - NIH Office of the Director

PROJECT SUMMARY Ion channels control the membrane potential and electrical properties of a myriad of excitable cells, making them critical components required for the physiological function of numerous organ systems including heart, brain, smooth muscle and skeletal muscle. The function of these channels and resulting electrical properties of the membrane are often evaluated using patch-clamp electrophysiology. However, as the number of variables underlying channel function has grown with increasing knowledge of normal and disease states, conventional manual patch-clamp methods have become increasingly insufficient to support the research. At the University of Maryland, Baltimore (UMB) our faculty includes a broad group of researchers focused on understanding the normal and pathophysiological mechanisms of ion channels and membrane electrical properties. The goal of this proposal is to support those scientists in their study of the electrophysiological characteristic of excitable cells, enabling enhanced mechanistic understanding of ion channel function and disease, and facilitating pharmacologic and therapeutic studies aimed at targeting ion channel function for therapeutic effect. This will be achieved through the purchase of an automated patch-clamp system, the Nanion Technologies SyncroPatch 384. The capability of the instrument to simultaneously patch up to 384 cells will dramatically increase the throughput of biophysical studies currently in process at UMB, enabling new resolution of genotype correlation with electrical phenotypes and promoting the development of new therapies. The Nanion SyncroPatch 384 is capable of recording in either voltage-clamp or current-clamp mode from a wide array of cell types, with data quality comparable to manual patch-clamp techniques, but with significantly greater throughput and reduced reliance on specialized user skills. The instrument will comprise the University of Maryland High -Throughput Ion Channel (HTIC) core and will be housed in the Department of Pharmacology and Physiology, where multiple faculty with electrophysiological expertise are available to support the instrument. Strong financial, administrative and facilities support will be provided by the University to ensure the success of the instrument, which will support a varied array of NIH-funded research projects. In summary, the SyncroPatch 384 offers significant advantages over conventional manual patch clamp techniques, including higher throughput, greater consistency and reproducibility, advanced temperature control, comprehensive data acquisition and analysis, versatility, and access to patch clamp techniques for a broader array of researchers.

OD - NIH Office of the Director

Summary: The Lundquist Institute (TLI) is requesting funds to purchase a Leica STELLARIS confocal microscope to be housed in its established, centrally managed core facility. This new system is intended to replace an aging 12- year-old Leica SP8 microscope that no longer meets the evolving needs of our research community. A broad user group of 12 investigators (10 of whom are NIH-funded), who are all making significant and pioneering contributions to cross- disciplinary research at the interface between developmental biology, cell biology, molecular biology, cancer, endocrinology, neurobiology, immunology, and host-pathogen interactions, will immediately benefit from the transformative imaging capabilities of the instrument. The STELLARIS system offers major advancements in confocal imaging technology, including a tunable white light pulsed laser for fluorescence lifetime imaging microscopy (FLIM), integrated with the high-speed FALCON FLIM platform and capable of multiplexing up to 11 spectral channels. These features provide users with quantitative imaging modalities to monitor complex dynamic processes in live and fixed samples. The instrument also includes LIGHTNING super-resolution capabilities based on adaptive deconvolution, expanded spatial coverage, and Leica's proprietary HyD detectors with tunable spectral sensitivity (1-nm precision, 400–850 nm), enabling high- resolution, low-phototoxicity imaging across a wide range of fluorophores. Acquisition of this system will ensure continued access to state-of-the-art imaging technology, enabling investigators to generate high-quality, multidimensional datasets and address increasingly complex biological questions. This instrument will directly enhance the rigor, reproducibility, and competitiveness of NIH-supported research at TLI by facilitating transformative insights into molecular and cellular mechanisms of health and disease.

OD - NIH Office of the Director

ABSTRACT/SUMMARY This proposal requests funds to purchase a Liberty Blue 2.0 solid-phase peptide synthesizer. At present, Vanderbilt lacks a comparable capacity for customized peptide synthesis, compelling researchers to rely on commercial vendors. While standard peptides can often be sourced at reasonable cost, the synthesis of peptides incorporating non-proteinogenic amino acids, macrocyclizations, or site-specific chemical modifications incurs prohibitive costs and prolonged lead times. These limitations negatively affect numerous NIH-funded research programs and severely lowers the chemical novelty accessible to investigators who make use of peptides in their research. Acquisition of an institutional instrument will directly address this gap, enabling timely and affordable access to high-quality, customized peptides that are increasingly central to modern biomedical research. This instrument will serve a large and scientifically expansive group of investigators across 15 departments in the College of Arts and Science, the School of Medicine Basic Sciences, and the Vanderbilt Institute of Chemical Biology. Investigators from the Vanderbilt University Medical Center will also have access. The user base spans a wide array of NIH-funded projects that rely on synthetic peptides. For example, one group synthesizes fluorophore-labeled peptides to monitor receptor trafficking. Another develops cleavable linkers that release antibiotics from antibody-drug conjugates designed to target methicillin-resistant Staphylococcus aureus. A third focuses on macrocyclic peptides that modulate the activity of CFTR and thus show promise as future therapeutics for cystic fibrosis. Several other groups engage heavily in structure- and AI-guided design and require rapid synthesis of candidate molecules to support downstream biochemical and cellular validation. The Liberty Blue 2.0, manufactured by CEM Corporation, uses microwave-assisted chemistry to accelerate synthesis cycles, improve coupling efficiency, and enhance overall yield and purity. The instrument accommodates a wide range of chemistries and scales, offering flexibility to support exploratory screening, structure-activity relationship campaigns, and early-stage preclinical development. Importantly, it also provides significant cost and time savings compared to commercial synthesis, especially for chemically complex sequences. The instrument will be housed within the Molecular Design and Synthesis Core, which has provided synthetic chemistry expertise and training to the Vanderbilt community since 2006. This core will oversee daily operation and user access, supported by administrative and financial contributions from the School of Medicine Basic Sciences and the College of Arts and Science. Acquisition of the Liberty Blue 2.0 will significantly enhance Vanderbilt’s infrastructure for chemical biology, lower the barrier to peptide-based experimentation, and accelerate discovery across multiple scientific disciplines and therapeutic categories.

OD - NIH Office of the Director

Project Summary/Abstract This application is to purchase an AxL Cleared Tissue LightSheet (AxL CTLS) from 3i for imaging of optically cleared samples. The Center for Advanced Microscopy is the only microscopy based core facility on the Chicago Campus and supports approximately 750 users from 300 laboratories annually. The Center for Advanced Microscopy has been supporting light sheet microscopy for 8 years with a Miltenyi UltraMicroscope II system. Light sheet microscopy has been very successfully incorporated into the workflows of many researchers at Northwestern University. Our UltraMicroscope II has been heavily used, but it has reached end-of-life and will no longer be supported by the manufacturer. The instrument is beginning to fail and in the past year we had 4 months of downtime for repairs. We seek to replace this instrument with a new light sheet that has higher resolution, faster speeds and can accommodate a wider range of samples. After testing several systems, we chose the AxL CTLS. Fifteen NIH-funded investigators are Major Users of this instrument and the success of their 26 NIH funded research is dependent on light sheet technology being available at CAM. Their research has a broad range of impacts across basic and applied science fields from fields including surgery, infectious diseases and microbiology, immunology, cardiovascular biology, developmental biology, neurosciences, and nephrology. Acquisition of the AxL CTLS would enhance the research of these investigators and offer improved image capabilities than the current instrument. The AxL CTLS would be a valuable acquisition not only for research carried out at Northwestern University and the Chicago scientific community.

OD - NIH Office of the Director

PROJECT SUMMARY We are requesting a 120kV transmission electron microscope (TEM) for the Electron Microscopy Core Facility (EMCF) at UT Southwestern Medical School in Dallas, TX. The EMCF is a campus wide, institutionally supported, fee for service facility that provides electron microscopy services for basic and clinical science investigators at UT Southwestern. In the fiscal year 2025 (which ended August 31, 2025) the EMCF served 132 users from 67 UTSW laboratories. Over the past twenty years, the EMCF has supported nearly 300 publications. The EMCF has two TEMs that together are used more than 3200 hours per year. The instrument we are requesting is intended to replace our nineteen-year-old Tecnai G2 Spirit Biotwin TEM that is near the end of its lifetime. The two cameras on the microscope are failing and are no longer supported by the vendor. The instrument itself has begun to experience significant down time due to the difficulty of finding compatible parts and is often only partially functional because of recurring issues. If this heavily used instrument fails, demand for the remaining microscope will significantly exceed the time available. Timely replacement of the Tecnai G2 will enable the health-related research of the 16 major users listed in this application as well as many other laboratories who use the EMCF in support of their NIH funded research. Among other projects, the requested microscope will be used to study the structure and function of insulin receptors, ion channels, the virulence of bacterial pathogens, lipogenesis, cardiac and skeletal muscle development, and the mechanism of amyloid aggregation. These studies will have an impact on our understanding and treatment of diseases such as diabetes, obesity, muscular dystrophy, cardio myopathy, Alzheimer's and Parkinson's.

OD - NIH Office of the Director

Twenty-four NIH-funded investigators studying a wide range of biomedical research topics at the University of Maryland Baltimore (UMB) are requesting funds for a transmission electron microscope (TEM), the Talos L120C G2 (Thermo Fisher Scientific). UMB has a long history of TEM use conducted at the Electron Microscopy Core Imaging Facility (EMCIF), and the decommissioning of the current TEM has drastically impeded the ongoing research projects. This TEM will replace an 20-year-old Thermo Tecnai T12, a 120 kV tungsten filament instrument, whose reliability and stability were decreasing when we lost the capability to capture images. Through demonstrations of microscopes from several vendors as well as Director and user experiences, the Thermo Talos L120C G2 is the ideal replacement research microscope with its high-contrast, high-resolution optics for biological samples. Its lanthanum hexaboride crystal electron source and Ceta 16M CMOS and Smart digital camera will produce bright, well-contrasted images for all our users. Its user-friendly software interface with features such as motorized stage, alignment recall, automated apertures, and rapid specimen exchange make this a good fit for a core facility. The remote operation features are ideal for training users and providing fee-for-service to distant users. The Talos L120C will be placed in the current TEM room, which is a pre-existing, well-designed space with convenient access to the main Core lab and staff. It will be administered by the EMCIF which is part of the Center for Innovative Biomedical Resources (CIBR) which provides resources to UMB researchers. Our staff will provide training in sample preparation and in operation of the new TEM. EMCIF is jointly supported by the School of Dentistry and the School of Medicine. We support research across the entire UMB campus as well as the University of Maryland System. The new TEM will be placed into the existing recharge system available to users through an iLab software interface available through the web and as a smart phone app. The new TEM will support continued progress of NIH-funded research projects of UMB scientists that are relevant to our mission of improving the human condition and serve the public good of Maryland. The EMCIF has a strong record of collaboration with researchers providing electron microscopy results for presentations, publications and grant applications. Strong institutional support provides for Director and staff salaries, space, instrument service/maintenance, administrative services and other EMCIF operating costs. If funded, the Talos L120C will be the primary TEM for UMB faculty research, and it will expand our capacity for biomedical research at UMB and at regional institutions. The impact will extend beyond the cancer, blood, infectious disease, bone development and other research projects listed in this proposal. The EMCIF’s mission is to provide state-of-the-art instrumentation and training to researchers, trainees, graduate students and staff. We aim to train the next generation of scientists to appreciate and interpret electron micrographs.