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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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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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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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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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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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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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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 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.

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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.

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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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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.

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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 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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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.

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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.

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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.

OD - NIH Office of the Director

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.

OD - NIH Office of the Director

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.

OD - NIH Office of the Director

The proposed comprehensive protein stability platform will serve multiple major users in assessing the properties of protein systems associated with immunomodulation, post translational modification effects, general receptor-ligand interactions, phase separation and condensates, medicinal chemistry screening, and translational therapeutics development. The proposed instrument combines full-spectral fluorescence monitoring, with dynamic light scattering (DLS) and static light scattering (SLS) with plate reading, sealable optical plates for thermal cycling, and full automation and data reduction. This will expand the assessment of stability and component interactions by the six major users and others for the NIH supported basic and translational research at Einstein funded through four Institutes. In the long term, the new available platform is expected to improve and expand the tools available for ligand screening, optimization of targets for structural biology investigations (XRC, NMR, CEM), and investigation of quaternary, quinary, and phase structural features in a systematic and reproducible manner in multi-channel mode on a single instrument. The instrument will be housed in a core facility with skilled and experienced management.

OD - NIH Office of the Director

We are requesting funds to purchase a single integrated system. A 4 color confocal microscope with resonant scanning capabilities on a motorized inverted stand with four GaSP detectors, two with customizable spectral tuning. This instrument will be housed within the Center for Biologic Imaging (CBI) at the University of Pittsburgh School of Medicine. The mandate of this core facility is to provide access to a full range of light and electron optical, image analysis, and morphometric methods to all research groups within the University of Pittsburgh School of Medicine. Point scanning confocal microscopy is an essential service provided by the center, and until last fall the core provided access to 6 different systems to our user base (>300 research teams and @1000 individual users). The average usage time for each of these systems is between 2,500 and 3000 hours a year and almost all the use is by groups supported by NIH grants. In September of last year the oldest of these systems, a 19 year old FV1000 (1S10RR022637-01) had multiple failures in the laser combiner which could not be repaired due to a lack of parts (see letter) and the microscope was retired. This January we were told that the oldest of our Nikon A1 confocals (16 years old) had multiple components which would no longer be covered by Nikon service due to a lack of parts (gas lasers and tubes and other hardware) (see letter) which means that future failure may make the system non-repairable and it should be replaced. The NIH funded research base in our institution continues to grow, and given the current use levels we feel our confocal capabilities are at saturation in fact the lack of available instrument time has become a major factor limiting the utility of the center, which in turn which has lead to considerable frustration amongst users. As pointed out by major users the requested instrument offers multiple technical advances over our current systems which motivate the specific instrument choice. However, beyond improved technology the absolute primary reason for submitting this application is to provide critically needed contemporary instrument time to a growing body of NIH supported users of the imaging core here at the University of Pittsburgh.

OD - NIH Office of the Director

PROJECT SUMMARY/ABSTRACT This proposal requests funds to acquire a LUMICKS C-Trap Dymo300 microscope to support NIH-funded investigators across multiple schools at Vanderbilt University. The C-Trap combines the ability to exert forces on molecules and cells through optical tweezers with the ability to visualize structures through confocal imaging with the sensitivity to detect single molecule fluorescence. The instrument is highly automated and capable of handling complex calibrations and work-flow scripts, facilitating access for a broader scientific community. The instrument also includes a microfluidic laminar flow platform that aids in assay construction and protocol refinement. This microscope will be located within, and maintained by, the Vanderbilt Center on Mechanobiology – an initiative that serves faculty labs within the School of Engineering, the School of Medicine Basic Sciences, and the College of Arts and Sciences. All three schools have provided matching financial commitments that will fully cover the service contract for the instrument. Multiple scientific thematic areas have been identified that will specifically benefit ~19 major and minor users from all three schools. Among these themes, Vanderbilt has a strong community of investigators focused on advancing our understanding of DNA repair, replication and gene regulation. These researchers will use the microscope to observe how purified multiprotein machines process and maintain the information encoded within DNA through binding, translocation, and remodeling events. A second theme supports Vanderbilt’s robust cytoskeletal and cell mechanics research programs. In this area, the C-Trap is ideal for visualizing and manipulating actin and microtubule filaments, along with their associated proteins and molecular motors. These studies will provide access to a whole new frontier for Vanderbilt investigators who can deeply probe complex associations among cellular machinery. A third theme will investigate the mechanobiology of T-cell receptor-antigen quality and T-cell activation, as well as other receptor- ligand associations and conformational motions. Additional areas of research involve membrane dynamics, biomolecular condensates, forces associated with phase separation and colloids, and studies of AAA+ protein machines that carry out a multitude of cellular processes. The microscope will serve as a regional resource and bolster teaching across all three schools. The C-Trap will be housed in a space where Vanderbilt has purposefully clustered mechanobiology faculty labs and positioned other core resources. Management, support of the microscope and projects, and long-term operation and maintenance of the system will be led by Dr. Matthew Lang, who has over two decades of experience in optical tweezers and single-molecule studies. Collectively, Vanderbilt is ideally positioned to make rapid and significant use of these new capabilities, extending our research scope across a broad range of NIH-sponsored studies.

OD - NIH Office of the Director

Project Summary This shared instrument grant requests funds for an Aurora CS for fluorescence-based cell sorting, to be installed at Yale University. Flow Cytometry is a critical resource for biomedical research. Nearly every discipline in the biologic sciences uses this technology that is critical for a variety of applications downstream of the sorting of cells. There is a growing demand for high-parameter flow cytometry at Yale to characterize biological samples as completely as possible. As the amount of published phenotypic information on distinct cell types and their subsets continues to increase, the number of parameters that is then needed to uniquely identify and distinguish diverse cell subset is likewise growing at a rapid pace. However, the number of cells within a sample can sometimes be small and as a result, an increasing number of studies require higher parameter panels during cell sorting to collect deep phenotypic information with small samples that cant also be analyzed separately at another instrument. For these reasons the Aurora CS is an ideal instrument to meet these needs because it 1) has full spectral detection with more than 60 sensitive detectors that generate spectral signatures that can be computationally “unmixed”, 2) enables measurement of over 30 cellular parameters simultaneously, 3) greatly improves the ability to distinguish multiple fluorescent proteins such as GFP, YFP or RFPs with overlapping emissions, or any other challenging dye combination, 4) identifies spectral signatures of unique cell-type specific autofluorescence that can then be computationally removed, solving a perennial problem with highly autofluorescent samples such as those derived from kidneys, lungs or intestines, and 5) further offers the ability to sort objects as small as 100 nm, such as EVs, synaptosomes or platelets. The requested equipment would replace a heavily used but aging BD FACSAria. The vendor will soon cease to offer service contracts on this model. Since this FACSAria is the only cell-sorting flow cytometer available in its building, any equipment breakdown could result in delays that would significantly impede ongoing research. It is critical that the existing instrument be replaced with a reliable cell-sorter that can be repaired in a timely fashion, preferably one that can be operated independently by investigators when staff is not available. The Aurora CS will serve as an ideal “future proof” cell sorter in a demanding location while addressing multiple other unmet needs on campus. Thus, the requested equipment will empower Yale researchers to interrogate complex biology with more challenging phenotypic panels, accelerating the pace of research while engendering greater insights and deeper knowledge.

OD - NIH Office of the Director

Project Description We request $450,305 to acquire a JEOL 400 MHz NMR spectrometer with a shielded magnet, 2-channel console, ROYAL HFX probe and autosampler to support cutting-edge synthetic chemical biology research at the University of Kansas (KU). The requested instrument will serve 15 highly active research groups, including three NIH-funded major users (Bloom, Farrell, and Sathyamoorthi) and 12 additional investigators, most of whom are also NIH-funded. This 400 MHz NMR will be housed in a long-established university-wide core facility with a strong track record for cost recovery, user training and instrument upkeep. The requested instrument will be integrated into the recently installed helium-recovery system, which will recycle over 85% of the liquid helium consumed during normal operation of the superconducting magnet and the gaseous helium expelled during periodic refills of the magnet dewar, significantly reducing the long-term operating costs and environmental impact. Based on current and projected needs, the spectrometer is expected to operate at 80% capacity. NIH-funded researchers will account for over 65% of total usage, with the three major users comprising more than half. This high utilization underscores the urgent need for a modern, reliable platform to support KU’s biomedical research. Currently, investigators rely on two aging 400 MHz NMR spectrometers, both over 20 years old, with obsolete components and growing maintenance challenges. Downtime and limited capabilities impose major delays and opportunity costs on research, despite the efforts of the highly skilled staff. Continued dependence on these outdated systems jeopardizes ongoing and future NIH- supported research. This proposal is driven by the critical need to sustain and advance KU’s synthetic chemical biology efforts. The new NMR spectrometer will directly support transformative research, including in a) the development of next-generation peptide therapeutics with enhanced stability, cell permeability, and reduced metabolic degradation; b) the design of novel chemical probes to identify neglected carbohydrate- binding proteins and better understand their roles in biology and disease; and c) the improvement of synthetic methodology for the preparation of novel anti-infective natural products to reduce human morbidity and mortality. The proposed instrument will be a vital asset to KU’s biomedical research enterprise, accelerating discovery and enhancing NIH-funded investigations in health-related chemical biology.

OD - NIH Office of the Director

PROJECT SUMMARY/ABSTRACT The CardioExcyte 96 is an advanced, label-free, impedance-based electrophysiology system designed for high- throughput, real-time monitoring of cardiac cell activity. Utilizing 96-well plates, it enables the non-invasive measurement of key cardiac parameters such as action potentials, contractility, and heart rate variability without the need for electrodes or dyes. This technology provides a powerful, scalable solution for assessing cardiac function, making it highly suitable for studies involving drug-induced cardiotoxicity, cardiovascular diseases, and diverse cell models. A major limitation in current cardiac research is the lack of platforms that combine high- throughput capability with detailed, non-invasive electrophysiological analysis. This gap has slowed progress in critical areas like drug safety testing, disease modeling, and personalized medicine. The acquisition of the CardioExcyte 96 will directly address this need by providing our institution with cutting-edge technology to enhance both the quality and efficiency of cardiovascular research. Integrating this system into existing workflows will support a wide range of investigations in cardiac electrophysiology, offering researchers the ability to generate high-resolution, real-time data that can accelerate discovery. The primary objectives of this project are to improve the accuracy and throughput of cardiac screening in drug development, advance studies on arrhythmias and cardiotoxicity, and foster collaboration across multiple research domains. Overall, the CardioExcyte 96 will significantly strengthen our institution's research infrastructure, enabling scientists to tackle key challenges in cardiovascular biology and therapeutic innovation more effectively.