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PSEG Foundation

Supports STEM education, workforce development, and community sustainability programs in PSE&G's New Jersey service area.

National Park Service

Linking college students and recent graduates to National Park Service sites and units for the purpose of completing STEM (science, technology, engineering, and math) internships in National Park Service units across the country.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Sequence-specific transcription factors (TFs) drive precise and timely control of gene expression during development. Changes in TF activity underlie much of the phenotypic divergence between species, and dysregulation is increasingly recognized as a cause of diverse human diseases. Despite the importance of TFs in cellular and organismal health and disease, most tools for studying TF function and conceptual models are binary in nature. The binary nature of TF models suggest an on-off switch, yet many disease-associated TFs operate in an analog fashion, where dosage is correlated with phenotypic outcome. Molecular tools to study and manipulate TF activities in an analog fashion are minimal, hindering quantitative modeling of TF function. This proposal seeks to fill this knowledge gap using an innovative chemical genetic approach that can precisely modulate TF dosage in human pluripotent stem cell (hPSC)-derived cell types. We will use this system to define how dosage of the key TFs SOX2, SOX9, and TWIST1 modulates chromatin state, gene expression, and cellular phenotypes and how this dosage-dependent regulation varies across cell types and evolutionary time. These TFs are ideal models to address our questions as they are associated with dosage-sensitive developmental disorders, function in multiple cell types, and have been associated with regulatory evolution. Our preliminary datasets validate our approach, revealing substantial variation in the dosage response of TF-dependent regulatory elements (REs) and genes, from highly sensitive to robust and buffered against quantitative TF dosage changes. We also recently applied cutting-edge deep learning models to learn sequence features underlying variation in RE dosage responses, revealing a surprising contribution of low-affinity binding motifs not apparent in unperturbed states. These results hint at a new layer of the cis-regulatory code, where certain sequence features are only apparent when studying transcriptional regulation in a quantitative regime. Our goal for this Program is to comprehensively characterize the underlying logic of these previously undefined additional regulatory layers and understand how they vary between cell types and species. To reach this goal, 3 directions will be pursued. 1) We will use multi-lineage differentiation of our unique hPSC reagents to investigate while certain cell types are sensitive to SOX2 and SOX9 dosage while others are buffered against quantitative changes. 2) We will apply chemical genetics and deep learning in chimpanzee PSCs to test whether the cis- regulatory logic defined in human cells generalizes across species and to distinguish cis from trans contributions. 3) Tool development, both experimentally where we will expand our system to allow bi-directional TF dosage control, and computationally, where we will develop improved deep learning models for predicting how REs respond to TF dosage. Together, these directions provide orthogonal but complementary approaches and tools that will converge on a unified model of how TF dosage impacts development and evolution, ultimately ushering our understanding of transcriptional regulation into a more mechanistic and quantitative realm.

U.S. National Science Foundation

Racial Equity in STEM Education Program Description (EHR Racial Equity) Persistent racial injustices and inequalities in the United States have led to renewed concern and interest in addressing systemic racism. The National Science Foundation (NSF) Directorate for Education and Human Resources (EHR) seeks to support bold,ground-breaking,andpotentiallytransformativeprojectsaddressing systemic racismin STEM.Proposalsshouldadvanceracial equity in science, technology, engineering, and mathematics (STEM) education and workforce developmentthroughresearch(both fundamental andapplied)andpractice. Core to this funding opportunity is thatproposalsare led by, or developed and led in authentic partnership with, individuals and communities most impacted bytheinequities caused by systemic racism. The voices, knowledge, and experiences of those who have been impacted by enduring racial inequities should be at the center oftheseproposals,includingin, for example: project leadership and research positions,conceptualization of theproposal,decision-making processes, and the interpretationand disseminationof evidence and research results. Theproposed workshould provide positive outcomes fortheindividuals and communities engaged and should recognize peoples humanity, experiences, and resilience.Proposalsneed to considersystemic barriers to opportunities and benefits, and how these barriersimpact access to, retentionin,and success in STEM education, research,and workforce development.Competitiveproposalswillbe clear with respect to how the workadvancesracial equity andaddressessystemic racism, as these constructs may have different meanings in different settings. Proposals should articulate a rigorous plan to generate knowledgethroughresearch (both fundamental and applied) and practice, such as, but not limited to: buildingtheory; developingmethods; testing approaches andinterventions; assessing the potential, efficacy, effectiveness, and scalability of approaches andinterventions; establishing,cultivatingand assessingauthenticpartnerships; changing institutional, organizational, and structural practices and policies; and/or focusing on affective, behavioral, cultural, social components,and implications. Contexts may include, but are not limited to: preK-12,two- andfour-year undergraduate, and graduate institutions; municipal organizations;STEM workplaces;andinformal STEM contexts, such as museums, community organizations, and media. In addition, proposals should include adissemination plan to proactively share what is learned with individuals and communities most impacted,as well asrelevant leaders, policy makers, and other stakeholders.Proposal budgets and project durations should be determined by the scope of the activities and in accordance with theNSF Proposal & Award Policies & Procedures Guide (PAPPG).PIs should include Racial Equity: at the beginning of the proposal title.

NIA - National Institute on Aging

The population of older adults (OA) is rapidly rising and anticipated to exceed 2 billion by 2050 causing an exponential rise in age-related comorbidities and healthcare costs. Age-related defects include mitochondrial dysfunction, inflammation, oxidative stress (OxS), insulin resistance (IR), genomic damage and endothelial dysfunction and result in declining physical function (gait speed and muscle strength), elevated blood pressure (BP) and higher waist circumferences. Via studies in OA and old mice (OM), we identified that deficiency of the body’s most abundant antioxidant Glutathione (GSH) plays a key contributory role for these defects in aging. GSH is an intracellular tripeptide composed of glycine, cysteine and glutamic acid, and declines with age. We found that GSH deficiency in OA occurs due to diminished synthesis caused by deficiency of glycine and cysteine (and not glutamic acid), and that GSH deficiency can be corrected by supplementing GlyNAC (combination of oral glycine, and N-acetyl-cysteine (NAC) as a cysteine donor because oral cysteine is absorbed poorly). In OM and OA, we discovered that GSH adequacy is critically necessary for efficient mitochondrial fuel (fatty-acid) oxidation (MFO) and for lowering OxS. In a small NIH-funded double-blinded, placebo-controlled, proof-of-concept pilot randomized clinical trial (RCT) in 24 highly selected, healthy OA and 12 young adults (YA) we reported that OA had (a) GSH deficiency in muscle and red blood cells; (b) impaired mitochondrial function; (c) deficient nutrient sensing; (d) increased inflammation; (e) elevated IR; (f) endothelial dysfunction; (g) genomic damage; (h) stem cell fatigue; and (i) cellular senescence. These abnormalities were associated with: (i) physical decline in gait speed, strength and exercise capacity; (ii) increased waist circumference; and (iii) higher blood pressure. GlyNAC (and not placebo) supplementation: (a) normalized RBC GSH concentrations, mitochondrial fuel oxidation, molecular regulators of energy metabolism, nutrient sensors, genomic damage, stem cells and cellular senescence; (b) lowered OxS, proinflammatory cytokines (IL6, TNFa, hsCRP); IR; endothelial dysfunction; (c) improved gait speed, strength, exercise capacity, body composition and systolic BP. GlyNAC supplementation in young humans had no impact. These data provide proof-of-concept that supplementing GlyNAC in OA corrects GSH deficiency and improves 7 aging hallmarks, and was not associated with any adverse effects. Could GlyNAC supplementation introduce a transformational change to improve the health of aging humans by promoting healthy aging? Although our completed RCT provides proof-of-concept for this, the sample size was small. Critically, the RCT was conducted in a rigorously screened cohort of healthy OA, using a high dose of GlyNAC. Therefore, it is important to definitively establish the validity and effectiveness of GlyNAC supplementation in a larger RCT conducted in a more typical population of OA, and also determine whether a lower GlyNAC dose, with lesser pill burden, could be effective. We propose a less invasive, less restrictive RCT in 150 more typical OA to determine the effects of supplementing GlyNAC on intracellular GSH, OxS, mitochondrial function, inflammation, IR, endothelial function, genomic damage, physical function, body composition and QoL. The proposed RCT will also test and compare two doses of GlyNAC to determine whether a lower dose of GlyNAC can be as effective as a higher dose on measured outcomes after 24-weeks.

NIA - National Institute on Aging

Project Summary We propose a multi-center randomized controlled trial (RCT) to test the effects of a perioperative geriatric intervention on postoperative outcomes for older patients undergoing cancer surgery, which will address the urgent need to improve operative outcomes for the geriatric oncology population. Major cancer surgery in older adults often results in serious adverse operative outcomes, leading to lengthy postoperative hospitalizations and diminished quality of life (QoL). These negative outcomes stem from the inherent operative risk combined with aging-related issues, such as multimorbidity, polypharmacy, frailty, and inadequate social support. Thus, perioperative interventions targeting the unique geriatric issues of older patients with cancer have tremendous potential to reduce surgical complications, shorten postoperative hospitalizations, and improve QoL. We will evaluate the Perioperative Intervention for Older Patients (PERI-OP) in a multicenter hybrid implementation-effectiveness trial. PERI-OP consists of the following: (1) a preoperative in-person or telehealth consultation with a geriatrician; and (2) postoperative inpatient geriatric co-management with the surgical team. We will enroll 354 patients scheduled to undergo major cancer surgery at two NCI-designated cancer centers and randomize them 1:1 to receive PERI-OP or usual care. We will test the effects of PERI-OP on postoperative health care use (e.g., days alive at home [primary outcome]), complications (e.g., delirium incidence), and patient-reported outcomes (e.g., QoL). We will utilize an effectiveness-implementation design to identify key facilitators and barriers to PERI-OP implementation and adoption. Aim 1: To assess the efficacy of PERI-OP on postoperative health care use and surgical complications. Hypothesis: The PERI-OP intervention will increase time alive and at home during the first 30 days after surgery (primary outcome) and will decrease postoperative hospital length of stay, intensive care unit days, hospital readmissions, delirium incidence, and surgical complications compared with usual care. Aim 2: To examine the impact of PERI-OP on patient-reported outcomes. Hypothesis: Participants assigned to PERI-OP will report higher postoperative QoL (30 days postoperatively as measured by the Functional Assessment of Cancer Therapy-General [FACT-G]), lower physical and psychological symptom burden, and better functional status compared with those assigned to usual care. Aim 3: To identify key facilitators and barriers to PERI-OP implementation and adoption. We will use a mixed-methods approach, guided by the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) and Consolidated Framework for Implementation Research (CFIR) frameworks, to explore how and why the intervention did or did not influence outcomes.

Raytheon Foundation

Major funder of STEM education programs for K-12 students and veterans transitioning to tech careers.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Intestinal stem cells (ISCs) maintain and regenerate the intestinal epithelium within a specialized niche known as the crypt. In mouse models, ISCs have been shown to activate a gene expression program similar to the developing intestine, referred to as ‘fetal reversion’ or ‘developmental reprogramming’ during injury repair/regeneration. Reactivation of this program is emerging as a critical process for effective regeneration. However, whether a similar developmental program is reactivated during human intestinal repair/regeneration remains unknown. The current proposal provides preliminary data that developmental reprogramming takes place during human intestinal repair/regeneration, and aims to interrogate the functional importance of development and repair mechanisms. As preliminary data, we have identified the EGF family member EPIREGULIN (EREG) as a developmentally expressed gene/protein that is re-activated after various forms of injury in vitro. In addition, we have developed novel iPSC-derived human intestinal organoid (HIO) injury models following transplantation into immunocompromised mice to create an in vivo-like injury and observe that EREG influences growth/proliferation in this transplanted HIO (tHIO) model. Based on our preliminary data, this proposal will test the hypothesis that the human intestine reactivates a developmental gene expression program following injury, including reactivation of EREG, which is an essential regenerative pathway. Using adult tissue-derived organoids and genetically engineered iPSC-derived HIO models, we propose three aims: (1) Define the developmental role of EREG through overexpression and knockout experiments in tHIOs; (2) Investigate the role of EREG during injury repair in EREG-modified tHIOs challenged with irradiation, 5-fluorouracil and Oxaliplatin; and (3) Elucidate the mechanistic pathway of EREG action, specifically evaluating downstream targets including c-MYC activation, that are crucial for the regenerative response.

NIGMS - National Institute of General Medical Sciences

Mechanical forces drive tissue function and pathophysiology, yet current high-throughput systems for drug development rarely incorporate mechanical forces, and those that do typically do not allow dynamic, feedback- based control over the forces acting on cells and/or engineered tissues. We propose to integrate key technologies developed by our team members: 1) rapid algorithms for directly estimating contractility of excitable tissues; 2) GPU-acceleration approaches for rapid computing; 3) externally triggered smart materials that can change their mechanical properties in response to magnetic fields; and 4) high-throughput engineered tissue platforms. This integration will allow us to create a high-throughput system that allows for real-time control over tissue mechanical loading based on the mechanical forces produced by the tissue. For this technology-development application, we propose milestone-driven efforts to optimize, validate, and integrate these technologies into a user-friendly, graphical-user-interface (GUI) supported platform. The approach we propose is unique in that the software-to-hardware interfacing, driven by imaging, can readily be adapted in the future by the research community, without requiring costly, user-dependent, one-time-use pure hardware-based approaches. The ability to parallelize the algorithm for computing tissue deformation, direct deformation estimation (DDE), will allow for dramatic acceleration of computing deformation, to the point that it can be computed in real-time, thereby allowing for magnetically-responsive biomaterials to be triggered in response to image-based data on contractility. We will demonstrate integration of our software-hardware interfacing based feedback approach by performing mechano-pharmacologic screens in skeletal muscle engineered from murine myoblasts and cardiac muscle engineered from human induced pluripotent stem cells. We will apply diverse loading regimes to the tissues, in combination with drugs known to have differential effects in mechanically loaded skeletal and heart muscle. We will also create tools for mining the resulting mechano-pharmacologic data. We envision that this technology will be broadly enabling for studies in mechanobiology and for improving translation of drug screens.

NIA - National Institute on Aging

Abstract: Stem cell exhaustion and related metabolic dysfunction are hallmarks of aging, compromising tissue regeneration/renewal leading to eventual loss of tissue integrity and function. Understanding metabolic programs necessary for proper stem cell function and aspects affected by age could open up therapeutic targets. Our initial metabolomics analysis in the muscle stem cell (MuSC) identified glutamine metabolism as a critical pathway during activation. Glutamine has multiple metabolic fates in mammalian cells. Aside from direct incorporation into protein, glutamine can be metabolized to generate nucleotides, other amino acids, and ATP via its anaplerotic metabolism in the TCA cycle in a “clockwise” direction. We have generated preliminary data using stable isotope-enabled metabolic flux analysis to demonstrate that both glutamine-driven oxidative and reductive or “counter-clockwise” pathways are active in muscle stem cells. Moreover, our data suggest that these pathways are impaired in the aged cell, in part due to decreased glutaminase expression. These preliminary findings lead to several new questions: 1- What are the relative roles of oxidative and reductive pathways of glutamine metabolism for energy generation and cell viability? 2-How does age alter these pathways, and does rescue activity restore cell function? We hypothesize that satellite cell function is dependent on glutamine metabolism via both the oxidative pathway for energy production and the reductive pathway, via isocitrate dehydrogenases 1 and 2 (IDH1/2), for generation of substrate for de novo lipogenesis (DNL) to ensure proliferative function and cell viability. Moreover, we hypothesize that impairment of these pathways contribute to age-related muscle stem cell dysfunction, which is reversible by restoration of glutamine metabolism. We will test these hypotheses through the following aims. Aim1 will test the hypothesis that glutamine-driven “bidirectional” metabolism is necessary for muscle stem cell function and myogenesis. We will use small molecule inhibitors of glutaminase and, as a specific manipulation of the reductive pathway, IDH2, to test this hypothesis. Aim2 will test the hypothesis that glutamine metabolism is impaired in old MuSCs, and that correction of this deficit, via viral or genetic glutaminase gain of function approaches, restores MuSC function and regenerative capacity to aged muscle. Aim3 will test the hypothesis that reductive glutamine metabolism fuels the DNL pathway to ensure cellular function. We will use heavy labelled flux analysis to determine how the reductive TCA pathway contributes to DNL and determine if lipid rescue can restore aged MuSC function. The proposed studies will fully test the hypothesis that glutamine metabolism is critical for muscle stem cell function, and that both oxidative and reductive TCA metabolism contribute to stem cell proliferation and survival. Moreover, given that the pathway appears dysfunctional in aging stem cells, our studies will test it’s potential as a therapeutic target.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

In the process of asymmetric cell division (ACD), two daughter cells with identical genetic information, but distinct fates, result from one cell division. Adult stem cells can undergo ACD to produce one self-renewed stem cell and one differentiating daughter cell. This process allows for important physiological processes including tissue development, homeostasis, and healing. Regulation of proper stem cell division can be lost in aging as well as cancer and other chronic diseases. How stem cells regulate ACD is not fully understood, but has broad implications for the study of cell fate determination and cellular reprogramming. To study ACD in an in vivo adult stem cell lineage, the Chen lab uses the Drosophila male germline. Germline stem cells (GSCs) in the Drosophila testis divide to generate both a self-renewed GSC and a differentiating daughter cell, called a gonialblast, which divides symmetrically to produce spermatogonial cells (SGs) that eventually undergo meiosis and terminal differentiation into sperm. Our group previously showed that histones that existed in the cell before DNA replication are retained in the self-renewed GSC, while newly synthesized histones are enriched in the differentiating daughter cell. The lab then discovered that during S- phase in GSCs, old histones are biased to the leading strand while new histones are biased to the lagging strand. Interesting, the catalytic subunits of both lagging strand enriched polymerase complexes, Polα and Polδ, are present at a lower level in GSCs as compared to SGs, while the leading strand enriched polymerase, Polε, shows comparable levels. Compromising Polα either pharmacologically (by an inhibitor) or genetically (polα+- flies), induces asymmetry in histone incorporation in SGs. However, how this differential expression of Polα and Polδ, but not Polε, is regulated is unknown. According to my preliminary data, polα and polε RNA levels reflect the protein level trend, and a stepwise increase in polα RNA across SG divisions was noted. I hypothesize that GSC transcriptionally repress polα, and this repression is lost as SGs divide. I will determine what cis-regulatory elements are responsible for this differential expression using a reporter assay. I will integrate transcription factor motif analysis, genomic data, and transcriptomic data to identify candidate trans-acting factors, which I can test further using molecular and genetic assays. In my second aim, I will investigate the functional outcome of reduced Polδ levels. The lab found that both polδ+/- and polα+/- flies display sustained fertility during aging. polα+/- flies also demonstrate enhanced regeneration in the germline. I will characterize the cell biology and morphology of polδ+/- testes during aging and test their regenerative potential using a genetic ablation experiment. The Chen lab has the expertise and resources necessary for me to carry out this proposed work. Between Dr. Chen’s mentorship and both academic and career support from the Johns Hopkins Department of Biology, I am confident I will have a successful doctoral research career and my findings will contribute to the NIH’s public health goals as well as benefit the broader scientific community.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY The endoplasmic reticulum (ER) performs many vital cellular functions, including protein and lipid synthesis and calcium storage, and importantly, the ER is a cell homeostasis sensor and signaling network. For example, when protein synthesis gets overloaded in the ER, the ER signals the integrated stress response. The ER also forms contacts with other organelles to signal and transport materials like calcium and lipids – ER health is therefore at the foundation of cellular health and disease prevention. In order to perform ER localized protein synthesis and signaling to other organelles, the ER network dynamically rearranges to form structures for dedicated functions. For instance, secretory cells are filled with expansive ribosome-studded sheets for protein synthesis, whereas cells which are constantly signaling, like neurons, are jam-packed with bundled tubules. So, what defines these functions and shapes? Changing ER protein levels alters both shape and function. I previously showed that ER-phagy, a process where ER membrane and attached proteins are selectively degraded, is required to set up the ER network as cells differentiate. Specifically, by deleting key autophagy and selective ER-phagy machineries in stem cells, I showed ER accumulation in axons and alterations in the ER proteome when they converted to neurons. These observations raise a key question: Are there similarities and/or differences in how these machineries function or are regulated in cell types with different functional outputs? In addition, we are missing key details about ER-phagy site formation and regulation, both under basal conditions in different non-dividing cell types and in post mitotic cells when stressors like antioxidant damage or protein misfolding occur. Thus, I will combine quantitative methods including ER protein turnover assays and mass spectrometry, with top-of-the-line imaging techniques including live-cell imaging and cryo-electron tomography, and functional assays to establish how ER clearance controls (i) the ER proteome landscape, (ii) ER structural rearrangements into tubules and sheets, and (iii) ER functions vital to cell-type establishment and homeostasis. In essence, the ultimate goal of my research program is to provide a blueprint detailing connections between the underlying ER proteome and how its regulation controls cellular function as cells specialize into unique types or respond to stress conditions. With extensive experience studying both ER-phagy and ER dynamics, I am well suited to achieve the goals of the proposed study, which lies at the intersection of the fields of organelle biogenesis and quality control. Defining the mechanisms driving the clearance of regions of the ER network and the specificity of these ER proteins in homeostasis and disease is essential to the field. In conclusion, the proposed work will lay the foundation for my laboratory, accelerating our research to the forefront of our field and deepening our understanding of key processes relevant to the areas of cell and developmental biology.

NIGMS - National Institute of General Medical Sciences

Abstract The Golgi apparatus is the central organelle in the secretory pathway, essential for processing and sorting of proteins and lipids, including receptors, hormones, and growth factors. In mammalian cells, multiple Golgi stacks are laterally interconnected into a continuous ribbon-like structure. Dysfunction and fragmentation of the Golgi ribbon are associated with various human diseases such as metastatic cancer, neurodegenerative disorders, or muscular dystrophies. Despite its essential function, the mechanism that regulate Golgi ribbon structure and function remain unclear. This lack of understanding stems primarily from limited knowledge of the molecules responsible for organizing and maintaining this structure. Our previous work showed that the ribbon structure depends on protein networks of Golgins; Golgi-resident membrane proteins defined by extensive coiled-coil domains that collectively form a matrix or scaffold. This matrix is crucial for both establishing the ribbon during interphase and for connecting Golgi membranes to the spindle during mitosis to ensure equal partitioning. However, the precise molecular mechanisms governing the organization of this scaffold or matrix remain unclear. In our recent effort aimed at identifying molecules that maintain the ribbon, we identified RNA an essential component of the Golgi matrix. The Golgin GM130 directly recruits RNA to the Golgi membrane to assemble a biomolecular scaffold or condensate that physically links Golgi stacks into a cohesive ribbon. Our central hypothesis is that RNA-Golgins assemblies function as a fundamental mechanism for organizing Golgi structure and function. We will employ a combination of biochemical, cell biological and microscopy approaches to elucidate the molecular mechanism by which RNA as a structural biopolymer scaffolds the Golgi ribbon. The objectives are to identify and characterize the dynamic organization of the RNA-Golgin scaffolds, and to define how this organization facilitates Golgi function at the molecular level. Furthermore, we aim to define the regulation of this scaffold to enable ribbon unlinking under stress conditions or during mitosis. Ultimately, this work will reveal the fundamental principles of how RNA as a structural biopolymer enables Golgi structure and function, define how cells use phase separation to organize organelle function, and provide novel mechanistic insights into neurodegeneration and cancer.

NIAID - National Institute of Allergy and Infectious Diseases

Our group recently identified a real-life model that uniquely illustrates the impact of the environment on asthma risk. Two populations of comparable Mexican ancestry living only 70 miles apart in Tucson, AZ, USA and Nogales, Mexico (MX) differ strikingly in their rates of childhood asthma, which are 4-fold lower in MX. The Binational Early Asthma and Microbiome Study (BEAMS-1, P01AI148104) was launched to test the hypothesis that MX or US residence impacts microbiome composition and metabolic capacity in pregnant mothers and their children, thereby shaping microbiome and immune responses and the child’s asthma risk. Initial findings from BEAMS-1 have supported this hypothesis. Asthma-predictive symptom (S) scores at 6-9 months (mo) were significantly higher in US than MX infants and were significantly associated with infant fecal microbiome composition and metabolome. Dietary lipids were the largest class of S-score-associated stool metabolites. Oleic, palmitic & linoleic acid metabolites were enriched in high S-score infants, while poly- unsaturated fatty acids were enriched in low S-score infants. These data linked dietary lipid stool metabolites to S-scores and infant asthma risk, but the underlying mechanisms remained unknown. Because asthma risk is associated with both early allergic sensitization and early respiratory viral infections, we now propose to define the mechanisms through which stool metabolites from BEAMS children with distinct S-scores differentially affect immune responses to airway viral infection and aeroallergens, and airway barrier integrity. Our plan stems from a rich set of preliminary data. Sterile fecal extracts (SFEs) from 1-mo-old infants with distinct S-scores 1) differed in their ability to reduce viral load and promote anti-viral responses in mice infected with human rhinovirus (RV), and 2) differentially affected aeroallergen-driven lung inflammation, in part through a novel B cell/IL17/neutrophil pro-inflammatory axis. 3) SFEs from 1-mo old MX infants more effectively promoted human airway epithelial barrier integrity compared to SFEs from their US counterparts. 4) Specific stool metabolites enriched in asthma-protected farm children protected mice from allergic inflammation and enhanced human airway barrier integrity. Therefore, we hypothesize that increased infant S-scores reflect the production of pathogenic microbial metabolites that dysregulate immune responses to respiratory viruses and aeroallergens, and impair airway barrier integrity, thus licensing the initiation of the asthma trajectory. Relying on extant BEAMS samples, we propose to expose mice to SFEs from 1- and 24-mo children with distinct S-scores and characterize the immune mechanisms and microbial metabolites that dysregulate responses to RV infection (Aim 1) and aeroallergen (Aim 2). (Aim 3) will characterize the transcriptional mechanisms that regulate human airway barrier responses to 1- and 24-mo SFEs from children with distinct S- scores, and their relation to SFEs metabolites. Our data will reveal mechanisms that connect gut microbial metabolites to asthma-predictive symptoms and inform approaches that target microbial metabolic dysfunction.

OD - NIH Office of the Director

PROJECT SUMMARY Neutrophils are critical for host defense against infection. Neutrophils can have either pro- or anti-inflammatory effects and exhibit significant heterogeneity and plasticity. There is a gap in understanding neutrophil heterogeneity and function because they are short-lived ex vivo and are not genetically tractable. Induced pluripotent stem cells (iPSC)-derived human neutrophils (iNeutrophils) offer the opportunity to genetically and developmentally program neutrophils for designed properties that may be used both to understand neutrophil biology and provide an avenue for engineering neutrophils for human disease treatment. We have engineered GMP-compatible human iNeutrophils that show improved antimicrobial function. Depleting the transcription factor GATA1 from human iPSCs results in a uniform mature population of iNeutrophils. However, the mechanisms of cell fate determination that result in neutrophil maturation and the specific role of GATA1 remains unknown. This neutrophil-biased differentiation potentially mimics the phenotype of neutrophilia seen in Down syndrome patients with a short isoform of GATA1 (GATA1s), suggesting the importance of full length GATA1 in regulating myeloid cell fates and functions in vivo. Here we propose to use single-cell multi-omics and imaging to understand how GATA1 and its isoforms modulate iNeutrophil development and function, with the long-term goal of increasing understanding of the drivers of neutrophil phenotypes to enable engineering of neutrophils with improved antimicrobial functions. We are uniquely positioned with the tools in place to make rapid progress with the proposed aims because of recent progress: 1. Our single-cell and metabolic imaging data show distinct neutrophil populations, with more maturity and less heterogeneity in GATA1 KO cells. 2. Preliminary data show epigenetic changes in the GATA1 KO that may indicate rewiring of gene networks in the absence of GATA1.

NIGMS - National Institute of General Medical Sciences

Project Summary: Cell migration is a critical aspect of normal development, as cells are often not born in the location of function, and migrate long distances to their final destination. But this process is often co-opted in disease states such as cancer. Much of our understanding regarding cell migration stems from work in 2D cell culture models, in which many types of cells exhibit a net forward movement through cell protrusion at the front and cell retraction at the back. Central to this form of migration is the ability of the cell to physically attach to its underlying substrate through the formation of focal adhesions. in vitro 2D and 3D approaches offer numerous advantages to dissect focal adhesion biology in terms of high-resolution image acquisition, control over matrix properties, and quantitatively assessing biophysical properties. While these studies have been immensely valuable, despite the years of research, it is still unclear what the organization and dynamic regulation of focal adhesions are during single cell migration in complex in vivo systems. Furthermore, pharmacological inhibitors have been developed against components of focal adhesion machinery for the treatment of disease without a clear understanding of how focal adhesions form and function during cell migration in animals. To meet this need, we developed a system in which we can directly visualize the formation and dynamics of focal adhesion structures of highly migratory single cells on a relatively planar surface of the zebrafish larval skin. Our recently published work challenges dogma established by in vitro studies, showing that phosphorylation of a key focal adhesion component, Paxillin, is greatly reduced in migrating cells in vivo, and that lack of Paxillin phosphorylation promotes focal adhesion disassembly rates and single cell migration in vivo, despite inhibiting cell migration in culture. This contradiction emphasizes the need for additional work in understanding focal adhesion regulation in complex in vivo environments (Aim 1). Furthermore, we found that the expression of the upstream kinase, FAK, is significantly reduced in cells in vivo compared to cells in culture. Given that FAK inhibitors are currently being developed in the clinic under the premise that part of FAK's function is for focal adhesion regulation, it is important to definitively determine whether FAK activity is required for focal adhesion regulation during single cell migration in vivo (Aim 2), and how Paxillin promotes cell migration in the absence of FAK phosphorylation (Aim 3). Identifying new proteins that interact with Paxillin in vivo will also begin to unravel why focal adhesion regulation is different in vivo versus in culture. Thus, our work has the potential to re-define the roles for focal adhesion proteins during single cell migration in vivo.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT CD8 T cells are important immune mediators central to protection against intracellular pathogens and cancer. Regulation of the delicate balance between T cell activation and quiescence is essential for mounting effective immune responses while preventing excessive inflammation. T cell dysfunction resulting from chronic antigen stimulation, known as “exhaustion”, leads to loss of protection against infections and cancer. In part related to this, chimeric antigen receptor (CAR)-T cell therapy has faced major challenges in eradicating solid tumors, with data demonstrating that the high antigen load contributes to chronic stimulation through the CAR, driving an exhausted phenotype. Conversely, an overactive and dysregulated T cell response can cause inflammatory disorders, including autoimmunity and graft-versus-host disease (GVHD), a deadly immune complications of hematopoietic stem cell transplant (HCT). Thus, effectively controlling T cell activation is key both to harnessing their potential to control infection and malignancies, and to managing pathologically inflammatory conditions. The transcription factor Hobit (ZNF683) has emerged as a novel central regulator for T cell activation and cytotoxicity. First identified as a lineage-specific marker of resident-memory T cells, Hobit has also been discovered across several cancer clinical trials to be expressed by exhausted anti-tumor T cells re-animated by PD-1 blockade. Hobit expression is lost in terminally exhausted T cells, suggesting a malleable role in T cell quiescence and re-activation. Compelling new work from our lab has deepened this paradigm shift: In the pivotal clinical trial that led to FDA approval of the CD28:CD80/86 co-stimulation blockade agent, abatacept, for GVHD prevention, we found that Hobit was significantly downregulated in the inflammatory proliferating T cells that normally precede acute GVHD (aGVHD), and that this downregulation was controlled with abatacept. Our goal is to uncover how Hobit governs T cell activation under chronic antigen stimulation and develop strategies to harness this pathway. We hypothesize that Hobit maintains antigen-experienced CD8 T cells in a state of revivable quiescence, which prevents terminal exhaustion and preserves their cytotoxic potential. To improve the outcomes of cellular therapies—by suppressing alloreactive T cells after HCT or enhancing persistence of CAR-T cells against tumor and antiviral T cells for chronic infections—it is critical to elucidate the molecular switches that control T cell cytotoxicity in the face of chronic antigen. We will approach this question through the following Aims: Aim 1: Determine if Hobit can preserve cytotoxicity in exhausted CD8 T cells. We hypothesize that Hobit can preserve cytotoxic potential in chronically stimulated CD8 T cells by inhibiting Blimp-1-mediated terminal exhaustion differentiation. Aim 2: Determine how Hobit expression regulates quiescence and cytotoxicity in transplanted CD8 T cells experiencing chronic antigen stimulation. We hypothesize that tuning Hobit expression can allow control over CD8 T cell activation during the chronic stimulation that occurs after HCT or after CAR-T therapy.

NCI - National Cancer Institute

TAL1 is a hematopoietic-specific member of the basic helix-loop-helix family of transcription factors (TFs) required for hematopoietic stem cell (HSC) function and the development of all hematopoietic lineages1-4. Aberrant activation of TAL1 oncogene associates with up to 60% of T-cell acute lymphoblastic leukemia (T-ALL) patients5-7. Its ectopic expression also led to development of leukemia or lymphoma in mice8,9. Deletion of TAL1 in T-ALL cells lost leukemic phenotype and induced apoptosis10. Furthermore, the TAL1 expressing (TAL1+) TALL subtype associates with poor prognosis and high rate of relapse indicating that dysregulation of TAL1 oncogene plays an important role in T cell leukemogenesis. In order to comprehensively understand the role of TAL1 domain structure in T-ALL pathogenesis, we employed unbiased sgRNA tiling library scanning of TAL1 exons for structural and functional domains in resolution of 3.9 base pairs (bp)/sgRNA. We identified that two lysine residues, K221 and K222, are critical for T-ALL cell function and survival. K221 and K222 sites of TAL1 are key targets for HAT mediated acetylation in vivo. The level of acetyl-TAL1 is lower in TAL1+ T-ALL cell lines than that of differentiated erythroid cells. Furthermore, we generated unacetyl-mimicking TAL1K221,222R (TAL1K2R) and acetyl-mimickingTAL1K221,222Q (TAL1K2Q) knock-in (KI) Jurkat cells and TAL1K2R and TAL1K2Q conditional KI (cKI) mice. While TAL1K2R Jurkat cells behaves similar to TAL1+ T-ALL cells that promote leukemic transcription profile for leukemia survival, TAL1K2Q cells facilitated T-cell differentiation and blocked leukemogenesis in xenograft mouse models, suggesting that acetylation status of TAL1 dynamically regulates TAL1 function in normal hematopoiesis vs leukemogenesis. In addition, IP-LC/LC MS revealed that TAL1K2Q mainly associates with HAT complexes while TAL1K2R interacts with HDACs and leukemic TFs. Based on these preliminary data, we hypothesize that acetylation status of K221 and K222 of TAL1 act as a molecular switch to control its ability to interact with TFs and epigenetic factors, as well as TAL1 mediated transcription circuit for normal hematopoiesis and T-ALL leukemogenesis. In this proposal, we will investigate: 1) key novel factors that are differentially associated with TAL1K2R and TAL1K2Q in controlling TAL1 transcription function in T-ALL; 2) the underlying mechanisms of TAL1 acetylation on TAL1-driven transcriptional regulation; 3) the biological function of TAL1 mutants in BM HSC regulation and T-cell leukemogenesis. To achieve this goal, we generated several mouse models of human T-ALL disease that are not only critical and essential to study how TAL1 acetylation alters HSC/LSC function during T-ALL development, but also potentially lead to the development of new therapeutic strategies targeting TAL1 modifications for T-ALL treatment. Success of the proposed studies, especially using our unique mouse models of human T-ALL disease, will lead to a better understanding of mechanism by which aberrant expression of TAL1 oncogene leads to T-ALL and how TAL1 acetylation status switches its ability to alter hematopoietic transcription regulatory networks in T-ALL.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

ABSTRACT Life begins with the fusion of unipotent sperm and egg, forming a totipotent zygote capable of generating an entire organism. The mechanisms governing totipotency during preimplantation development remain poorly understood. RNA modifications, such as methylation, play essential roles in post-transcriptional gene regulation. Here, we aim to explore the role of a putative RNA methyltransferase in regulating totipotency and early embryonic development. The zygote is initially transcriptionally silent, relying on maternal RNA and proteins. Its genome undergoes transcriptional activation through zygotic genome activation (ZGA), a pivotal process involving two phases: minor ZGA and major ZGA. This transition is characterized by a dramatic increase in gene expression, enabling further embryonic development. RNA modifications, including methylation by METTL proteins, play key roles in gene expression. Analysis of single cell sequencing data from mouse preimplantation embryos revealed that Mettl7a1, encoding a putative RNA methyltransferase, is highly expressed during 1-cell and early two-cell stage, coinciding with minor ZGA. Knockdown of Mettl7a1 in mouse embryonic stem cells (mESCs) downregulated totipotency markers and reduced the proportion of 2- cell-like cells (2CLCs), which exhibit features of totipotency. Based on these observations, we hypothesize that Mettl7a1 regulates ZGA and totipotency via its RNA methyltransferase activity. Aim 1 will investigate Mettl7a1’s role in minor ZGA and preimplantation development. Aim 2 will identify RNA targets and modifications catalyzed by METTL7A1. This work will uncover novel mechanisms of totipotency regulation, advancing knowledge in developmental biology, regenerative medicine, and epitranscriptomics.

NCI - National Cancer Institute

Project Summary. For proteins implicated in human diseases such as cancer, therapeutic advances arise not only from discovering ways to manipulate their function directly, but also through indirect approaches such as investigating their regulation by other cellular molecules. Heterotrimeric G proteins of the G12/13 subfamily provide a good example; the GTP-binding a subunits - Ga12 and Ga13 - are substrates for several chemical modifications after their translation. Two such mechanisms, covalent attachment of a specific fatty acid (S- palmitoylation) and a phosphate group (phosphorylation), were reported soon after discovery of these G proteins; however, follow-up studies of these modifications have been sparse. The specific enzymes that covalently modify the G12/13 a subunits have not been identified. Because Ga12 and Ga13 harbor potent ability to drive tumorigenic signaling and metastatic invasiveness in cells, and recently were implicated as conferring stem cell-like properties upon tumor initiating cells, methods of disrupting or manipulating their signaling could lead to useful therapeutics. My laboratory's undergraduates used mutagenic strategies to either nullify or mimic phosphorylation of Ga12 and Ga13 at selected amino acids, and recently obtained experimental results for Ga12 that point to a rarely observed cross-regulatory mechanism between its phosphorylation and S-palmitoylation. This putative mechanism, in which down-regulated S-palmitoylation of Ga12 disrupts its ability to drive tumorigenic signaling, has not been reported for any G protein. The first phase of this proposal describes a plan to “dissect” this cellular mechanism by i.) identifying enzymes that catalyze S-palmitoylation and phosphorylation of Ga12, ii.) revealing the contextual amino acids in Ga12 that allow its specific targeting, and iii.) developing assays to detect the S-palmitoylation and/or phosphorylation of recombinant Ga12 mutants expressed in cultured human cells. The second phase of this proposal also arose from a discovery by UNC Asheville undergraduates: a new mechanism in which activation of protein kinase A (PKA) drives a steep increase in the already potent tumorigenic signaling by both Ga12 and Ga13. A combination of mutagenesis, signaling assays, and molecular probes will be employed to investigate the components of this regulatory mechanism. Interestingly, the responses of Ga12 and Ga13 to PKA activation show several differences that will be investigated. An overarching goal of the proposed studies is to discover distinct features of tumorigenic signaling by the G12/13 subfamily, not only in comparison to other G protein a subunits but between Ga12 and Ga13 themselves. Such information should provide value toward developing therapeutics against cancers in which aberrant signaling by a specific G protein plays a role.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Project Description: Viruses are obligate intracellular parasites, as they require access to cellular translation machinery for viral protein synthesis. The order Mononegavirales contains non-segmented negative sense RNA viruses (nsNSV). These viruses transcribe positive sense, capped and polyadenylated mRNA that are indistinguishable from host transcripts. Due to this, it is assumed that these viruses use canonical translation machinery. However, the mRNA of these viruses possesses characteristics, such as extremely short 5’UTRs and strong stem-loop structures, that would preclude canonical translation initiation and scanning methods. Using a series of auxin-inducible degron (AID) systems, we have systematically probed the requirements of individual translation factors for protein synthesis. Vesicular stomatitis virus (VSV) is a member of the Mononegavirales order. Its translation has been the most studied of the nsNSVs. We have demonstrated that VSV mRNAs translate independently of factors necessary for canonical translation, leading us to believe that viral translation occurs through an alternative mechanism. This proposal aims to characterize the mechanism of VSV translation, a first step to expanding the knowledge of nsNSV translation. Long-term, this work will contribute to increasing the basic understanding of viral replication as well as identifying targets for the development of antivirals. Training Plan: I will receive guidance and feedback from my sponsor, Dr. Shawn Lyons, and co-sponsor, Dr. John Connor. In the lab, I will develop and refine my skills in biochemistry, translational research methods, and virology. Participation in regular lab meetings and journal clubs will hone my scientific and critical thinking skills, while attendance at departmental seminars will broaden my understanding of biochemistry and cell biology. Furthermore, I will gain expertise in RNA biology through monthly RNA club meetings, providing a specialized area of focus. I will regularly exercise my presentation skills, presenting in various fostering effective scientific communication skills. This comprehensive training will equip me with a strong foundation in biochemistry, virology, and RNA biology, positioning me for success in my current research endeavor. Further, this will provide me with the expertise necessary to obtain a postdoctoral training fellowship, which will guide me toward my ultimate goal of becoming a principal investigator in an academic setting. Environment: The Lyons lab is well-equipped with ample bench space, with various machines at our disposal. These include a Chemidoc and TurboBlot Transfer system, for western blotting, and a BioComp, for polysome analysis. We have access to shared spaces that accomadate cold storage and tissue culture. Within the building, we have access to several microscopes and a BioRAD NGC chromatography system, for protein purification. Additional equipment is available through various core facilities on Boston University’s Medical School and Charles River campuses.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

PROJECT ABSTRACT Mesenchymal stem/stromal cells (MSCs), the most abundant adult precursor cells in the human body, have been widely explored for various experimental and clinical applications and were recently approved for clinical use by the FDA. Nevertheless, a daunting challenge remains: the poor survival and reduced regenerative capability of MSCs in hostile microenvironments in the recipient tissue markedly undermine their efficacy and treatment outcome. Our proposal, “Reinvent Mesenchymal Stem Cells - A Synthetic Biology Approach to Engineer Programmable Adult Stem Cell Theranostics,” aims to overcome these limitations by engineering a programmable cellular theranostic platform for MSCs. We previously developed synthetic genetic circuits (SGCs) having upstream genetic sensors to detect intracellular signaling and downstream secretory effector molecules to program cellular signaling and desirable cellular functions under conditions of stress or disease. Here, we propose to integrate SGCs into MSCs to engineer Autonomous Impairment-inducible MSCs (aiMSCs). aiMSCs would spontaneously detect stress- or disease-induced intracellular signals via genetic sensors and initiate transgene expression to enhance cell survival and treat targeted pathologies. To ensure the safety and translational potential of aiMSCs, we will embed two types of autonomously programmable control mechanisms: 1) dual intracellular signaling feedback controls at the sensor and effector levels, and 2) a safety switch based on endogenous microRNA binding, to respond to the microenvironment in real time and increase effector output levels when the tissue damage intensifies or turn off the SGC when the tissue heals. As proof-of-concept, we propose two specific aims to develop the aiMSC technology and explore its potential as a next-generation MSC therapy. We will use synthetic biology, genetic engineering, and computational tools to engineer: 1) murine bone marrow (BM)-derived MSCs that automatically detect and respond to inflammatory signals, augment MSC survival, and promote a destructive-to-constructive transition in tissue inflammation (aiMSC-I); and 2) human BM-MSCs (hMSCs) that detect and respond to metabolic distress- induced signals and promote hMSC survival, anti-oxidation, and mitochondrial protection (HaiMSC-M). We will use cellular, organoid, and animal disease models to validate aiMSC-I and HaiMSC-M and to demonstrate enhanced MSC survival and functioning, as well as the self-contained and self-regulated modulation of disease conditions. Large-scale genetic sensor screening and machine learning will be used to identify unique MSC- specific pathology sensors. aiMSCs represent an adaptive, self-programmed theranostic approach that augments MSCs on demand in a safe and controlled manner. aiMSCs would significantly advance patient treatment and offer new perspectives for precision medicine, designer therapeutics, and stem cell therapies.

NIA - National Institute on Aging

Abstract Hematopoietic stem cell (HSC) transplantation (HSCT) and HSC based gene and cell therapies hold curative potential for diverse hematologic and immunologic disorders. However, donor age remains a critical determinant of transplant success. Aging drives profound HSC dysfunction, manifested by diminished regenerative potential, lineage skewing toward myelopoiesis, and an increased burden of clonal hematopoiesis (CH) associated mutations. These defects heighten the risk of graft failure, donor-derived leukemia, and poor survival after HSCT. Aging-associated HSC decline is driven in large by mitochondrial dysfunction. Aged HSCs exhibit impaired mitophagy, aberrant oxidative phosphorylation, and disrupted bioenergetics, changes that are further amplified by chronic inflammation and remodeling of the bone marrow (BM) niche. The fate and function of HSCs are shaped not only by their intrinsic metabolic state but also by their communication with niche partners, particularly mesenchymal stromal cells (MSCs). Our recent work demonstrated that HSPC-BM niche interactions are metabolically coupled via Connexin-43 dependent mitochondrial transfer, preserving HSC survival and niche function. Moreover, targeted mitochondrial supplementation into HSC preserves mitochondrial integrity, enhances metabolic adaptability, and maintains self-renewal during stress hematopoiesis, highlighting the importance of both mitochondrial quality and intercellular mitochondrial communication in hematopoietic regeneration. Our preliminary data show that mitochondrial transfer from HSPCs to MSCs markedly declines with age, compromising MSC bioenergetics and weakening their ability to sustain balanced hematopoiesis. We further identified that supplementation of aged HSCs with young mitochondria rescues mitochondrial membrane potential, restores AcH4K16 polarity, and rejuvenates transcriptome of aged HSC. Together, these findings highlight mitochondrial quality and transfer as central drivers of hematopoietic aging and compelling therapeutic targets. This proposal seeks to define the mechanistic basis and therapeutic potential of mitochondrial engineering to rejuvenate aging hematopoiesis. In Aim 1, we will test whether heterochronic mitochondrial supplementation and augmentation of mitochondrial function restore the youthful potential of aged HSCs by rewiring metabolic flux, enhancing autophagy, and resetting epigenetic programs. In Aim 2, we will dissect how HSPC-MSC mitochondrial transfer governs niche metabolism, trophic function, and the competitive fitness of aged versus CH-mutant HSPCs. By integrating metabolic, epigenetic, and intercellular communication pathways, this work addresses fundamental drivers of hematopoietic aging. The anticipated outcomes will provide mechanistic insight into how restoring mitochondrial quality and transfer can normalize aged HSC function and niche support. Ultimately, this study lay the foundation for innovative, clinically translatable strategies to improve HSCT outcomes in older patients and donors, while reducing risks of clonal dominance and age associated hematologic disease.

NIH

For decades, orthopaedic endoprostheses have been manufactured using traditional machining techniques. Specifically, the stem of the endoprostheses would be fabricated using mills and/or lathes, then the osseointegrated (OI) region would be added onto the stem using small metal beads, then sintered for strength. Recently, additive manufacturing, also known as 3-dimensional (3D) printing, has been introduced to provide affordable devices for limited markets. Instead of the traditional multi-step machining process, both the endoprosthetic stem and the OI region are designed to be manufactured in one continuous process, saving valuable resources. While there are FDA-related issues to address before 3D-printed devices become commonplace in the US healthcare system, many short-term implanted medical devices like orthopaedic fixation plates, screws, pins and wires, and even some long-term dental devices like dental fillings, crowns, and dentures are now 3D-printed. However, 3D-printing of many devices, including percutaneous OI endoprosthesis, is not yet widely accepted by the FDA because of the lack of data verifying that the device designed meets the engineering requirements, and validating that the device produced meets the need for which it was designed. Because of this, 3D-printing manufacturers are apprehensive to put their devices directly into humans. Fortunately, our research team has a well-developed sheep forelimb amputation model which has been directly used for this application and has been accepted previously by the FDA as a valid, preclinical model. In this proposed study, we plan to replicate the existing human Percutaneous Osseointegrated Docking Systems (PODS) endoprostheses to develop a 3D-printed version for use in sheep, which we will call sPODS, an acronym for Sheep PODS. The sPODS will be manufactured not only using a 3D-printing process, but also using a traditional machining process to act as a baseline for direct comparison. Both the 3D-printed and the traditionally machined sPODS will have the same external geometry (length, diameters, tapers, etc.), with the only difference being the method used to create the device. Once manufactured, the 3D-printed and the traditionally machined sPODS will be verified through benchtop testing, measuring the amount of bone loss during implantation, the contact area between the bone and the endoprostheses, and the fixation strength post-implantation. This will verify that the device manufactured meets the specified engineering requirements. Next, production quality 3D- printed and traditionally machined sPODS will be surgically implanted into our amputated sheep metacarpal model and validated against our published historical sheep data, confirming that the device produced meets the need for which it was designed. This research project with the following Specific Aims: Specific Aim 1: To quantify the surface, material, and mechanical properties of the 3D-printed sPODS and the traditionally machined sPODS, then to measure and compare the initial tensile and torsional stabilities of devices implanted into sheep carcass metatarsals. Specific Aim 2: To validate that the bone morphology and the tensile and torsional stability of the 3D-printed sPODS are not inferior to traditionally machined sPODS following 6-months in situ in a sheep amputated metacarpal model. Prior to implementing PODS clinically, this study is essential to confirm that device design aligns with engineering and material requirements. Ultimately, these efforts will immediately contribute to enhancing functional independence and quality of life of the Veterans with transhumeral amputations but will also serve as guidance for all teams designing and testing 3D-printed percutaneous OI devices at any anatomic site.