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OD - NIH Office of the Director

ABSTRACT Millions of people in the US are impacted by gastrointestinal diseases including Inflammatory Bowel Disease (IBD), Metabolic Disfunction Associated Steatotic Liver Disease (MASLD) and Pancreatitis. There are only a small number of drugs for IBD and MASLD, and none for Pancreatitis, making this a critically significant clinical question. Animal models have proven inadequate surrogates for these diseases and reliance on current preclinical evaluations are considered to be among the most problematic steps in drug discovery. The goal of Cincinnati Advanced NAM Development and Operational Research center (CANDOR) is to develop combinatorial New Approach Methodologies (NAMs) that more accurately model the pathophysiologic complexity and drug responses in patients with these gastrointestinal (GI) diseases. We have established an interdisciplinary team of collaborators of clinicians, scientists, experimental and computational biologists with a history of developing in vitro organoid and in silico NAMs with a focus on inflammatory diseases of the GI tract. CANDOR will provide a collaborative pipeline starting with existing cohorts of deeply phenotyped patients with IBD, MASLD, and Pancreatitis. Clinical data and patient samples will be used to build in silico NAMs, based on molecular pathways and cell-cell interactions that corelate with patient outcome and drug response. Each disease will have a corresponding in vitro NAM comprising intestinal, liver, and pancreatic organoids each with immune cells. Pluripotent stem cell banks have been generated from patients with each of these diseases, and healthy controls, and all organoid platforms are established and benchmarked to human samples. The aims of CANDOR are to establish in vitro NAMs that accurately model clinical features of IBD, MASLD, and Pancreatitis; to build disease-focused in silico NAMs that are based on gene regulatory, cell-cell interactions, and pharmacometric models from patients and combine these with data from in vitro NAMs; and to validate and disseminate combinatorial NAM technologies through training, outreach, and distribution.

U.S. Mission to Poland

Purpose of Grants: PD Poland invites proposals for programs that strengthen ties between the United States and Poland through activities that highlight shared values, promote bilateral cooperation, and forge enduring connections between the United States and emerging Polish leaders (high school students, university students, and young professionals ages 16 to 35), as well as established community leaders in the public, private, and nonprofit sectors. All proposals are required to have a clear connection to the United States, either through U.S. organizations, experts, and/or best practices in order to increase the awareness and understanding of U.S. perspectives, policies, and society. Proposals without significant U.S. content will not be considered for funding. Examples of possible public diplomacy grant activities include, but are not limited to: Youth engagement programs. Participatory and/or problem-solving workshops like tech camps. Soft skills and leadership-building workshops, seminars, and trainings that develop human capital and social or economic innovation. Workshops, seminars, trainings, master classes, and exhibitions on themes or topics that advance shared democracy, economic, and security goals. Programs that reinforce and amplify lessons learned by Polish alumni of U.S. Government-funded and private sector exchange programs. Priority Program Areas: ECONOMIC PROSPERITY Addressing barriers to the advancement of women in STEM fields and business. Strengthening the business skills of young entrepreneurs. Sharing best practices of U.S. businesses operating in Poland. Promoting the development of trade and investment with the United States, including entrepreneurship, small- and medium-sized businesses, and innovation as the basis for strong, sustainable, inclusive economic growth that creates quality employment and incorporates diverse and excluded groups. Promoting joint Polish-U.S. science, space, and innovation initiatives carried out by research organizations, nongovernmental organizations, universities, and private companies. ENSURING SECURITY Demonstrating the benefits of the of the Polish-U.S. security partnership and NATO Alliance for Polish emerging leaders (high school and university students ages 15-25 and/or young professionals ages 25-34). Promoting a deeper understanding of the impact of Polish and U.S. political, military, and humanitarian support for Ukraine and for Ukrainians in Poland. Strengthen cyber security awareness. STRENGTHENING DEMOCRACY Leadership training fostering innovation and critical thinking among young people (ages 16 to 24). Strengthening media practitioners and media consumers media literacy and ability to detect and combat mis/disinformation. Promoting Holocaust education and/or human rights education. Participants and Audiences: Proposals should describe both the primary and secondary audiences for the program, including anticipated numbers to be reached. Primary audiences are those who will participate directly in the program, while secondary audiences are those who will be reached by the project s primary audiences as a result of their participation. Priority target audiences in Poland for this funding opportunity are youth and young professionals (aged 16 to 35) who have demonstrated strong leadership potential, established professionals engaged in fields relevant to the U.S.-Polish partnership, and community leaders. The following types of programs are not eligible for funding: Programs relating to partisan political activity; Charitable, clinical (including mental health services), or development activities; Construction programs; Programs that support specific religious activities; Fund-raising campaigns; Lobbying for specific legislation or programs; Academic or scientific research; Programs intended primarily for the growth or institutional development of the organization; and Individual travel to attend a conference and/or courses at any educational institution. This funding opportunity aims to support specific projects with objectives that can be achieved within a set timeframe. We will not accept applications that are aimed more broadly at supporting your organization s usual or typical daily activities and operations. Those will be deemed technically ineligible and will not be considered for funding by the review committee.

FDA - Food and Drug Administration

Abstract Acquired aplastic anemia (AA) is a life-threatening disorder caused by an autoreactive T-cell mediated destruction of hematopoietic stem cells resulting in the inability to produce adequate red blood cells, white blood cells and platelets. Acquired AA is extremely rare, occurring in 2-6 patients per million. There are between 600- 900 new cases each year in the United States. While AA can occur at any age there is a bi-modal distribution with peaks in late childhood/early adolescence and in older adults. Patients with AA are susceptible to potentially fatal opportunistic infections, clonal hematopoiesis/leukemogenesis, and chronic transfusion burden. The workup of a patient with suspected AA takes several weeks during which time the patient receives only supportive care. Immune suppression therapy (IST) and bone marrow transplant (BMT) are the two therapies available for patients once a diagnosis is definitive. For patients with an available matched related donor (MRD), BMT is the standard of care (SOC). Patients lacking a MRD traditionally received IST although many institutions are now prioritizing alternative donor transplant. IST has a 50% response rate over time with the other half of patients requiring additional therapy. BMT has a higher disease-free survival rate but increased potential toxicities including graft versus host disease, infertility and graft rejection. The decision of which therapy to pursue is often the most anxiety-provoking time for families with children that have newly diagnosed AA. This Phase 2a/2b Trial Emapalumab: A Window of Opportunity in Pediatric Aplastic Anemia leverages data showing that the Interferon-gamma (IFNγ) pathway is associated with the pathogenesis of AA. Pediatric patients with newly diagnosed AA will receive a prophase of an IFNγ neutralizing monoclonal antibody called emapalumab. This prophase will not add time to curative therapy and will occur during the workup period between presentation and start of definitive therapy. At the conclusion of the prophase, patients that have a hematologic response will be consolidated with IST while those that do not will receive institutional SOC. In this way we create an algorithm to try and identify patients that are most likely to have a favorable response to IST. This data-driven identification of which patient should receive IST will help alleviate parental anxiety in making these decisions without sufficient information. Aim 2 of this project seeks to extend our previous findings that distinct patterns of pediatric clonal hematopoiesis are associated with poor outcomes after IST. Conversely, lack of these markers aligned with favorable IST responses. We will prospectively validate these findings and assess if these specific clonal changes can be used as predictive biomarkers for response to IST. We will also examine if an early upfront prophase with emapalumab can prevent and/or minimize emergence of clonal hematopoiesis by preserving larger reservoirs of hematopoietic stem cells. By combining the data from these two aims, we hope to provide an algorithm to identify pediatric patients with aplastic anemia that are most likely to be cured by IST.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY The leap from 2D cell culture to functioning 3D organs is one of the biggest opportunities in developmental biology and patient-specific tissues/personalized medicine. Our current method for growing complex 3D microphysiological systems (MPSs) relies heavily on the innate biology of adult induced pluripotent stem cells (iPSCs) to differentiate into organ-specific cell types, gently guided by the addition of soluble morphogens that direct differentiation. Unfortunately, the result of such a biology-driven, uncontrolled process are small organoids that lack reproducibility, specificity in cell type composition, and functionality. They exhibit uncontrolled size, heterogeneity of shape, and lack appropriate vascular, immune, neural components and organ-specific morphological features. To address this challenge, we need “smart”, stimulus responsive systems that offer control over various facets of cell growth and differentiation. This program’s goal is to develop smart, photoresponsive biomaterials to control and direct biological events, which will allow the study of more complex tissue environments and development of biologically relevant microphysiological systems. Directing the growth of 3D tissues requires spatial and temporal control across multiple factors, including matrix density, porosity, and gradients of soluble morphogens. Smart biomaterials that incorporate a light-triggered response offer the best level of control over these factors. Current photocleavable chemistries (chromophores that undergo bond cleavage when a photon is absorbed) are synthetically challenging to access, which has stalled their application across diverse material supports. We need a simple, readily modified chromophore that is material-agnostic for ready translation into any material support needed across this diverse field. Our unique approach to develop material agnostic photocleavable crosslinkers builds upon the powerful photochemistry and ready synthesis of ruthenium polypyridyl complexes. We have already shown excellent biocompatibility, synthetic flexibility with multiple reactive chemistries, and excellent photophysical properties of Ru complexes in biomaterial systems. This program will leverage this chemistry and demonstrate its broad application across multiple systems that provide spatiotemporal control of physical and biological cues in two main projects: 1) probing pathophysiological systems through control of the physical extracellular environment, and 2) directing cell attachment and growth through spatiotemporal patterning of biochemical cues within a material.

NINDS - National Institute of Neurological Disorders and Stroke

Human induced pluripotent stem cell (hiPSC) differentiation offers a unique perspective on species- specific aspects of neuronal development. We employed high-temporal resolution single-cell expression analysis to investigate the mechanisms underlying prolonged and enhanced neurogenesis in the human spinal cord compared to mice. Canonical correlation analysis revealed "human-specific" progenitor clusters marked by early co-expression of NKX2-2 and OLIG2. Lineage tracing revealed that these cells are bone fide motor neuron progenitors. Unlike classical motor neuron progenitors (pMNs), these more ventral motor neuron progenitors (vpMNs) exhibit increased NOTCH and WNT activity, generating motor neurons in a delayed and protracted manner. Furthermore, vpMNs undergo more rounds of cell division, yielding approximately five times more motor neurons that are enriched in motor neuron subtype innervating limbs. Evolution of a new progenitor domain is a novel mechanism through which human CNS increases its size and complexity, distinct from transit amplifying progenitors described in the developing human neocortex. Our proposed research aims to answer four outstanding questions: 1) Is NKX2-2 expression both necessary and sufficient to activate the vpMN program, leading to extended motor neuron genesis? 2) What evolutionary changes in the OLIG2 regulatory system allow human-specific co-expression of OLIG2 and NKX2-2? 3) Does increased NOTCH signaling observed in vpMNs contribute to their specification and delayed neurogenesis? 4) Do vpMNs and pMNs generate different subtypes of motor neurons during human neurogenesis? Addressing these questions will provide valuable insights into the molecular and cellular mechanisms that contribute to the increased number and complexity of motor neurons produced during the human spinal cord development. These insights might lead to improved motor neuron disease models that recapitulate more faithfully human pathology.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Mechanical forces are essential for neural development, and their dysregulation leads to neurodevelopmental defects. The mechanically-activated ion channel PIEZO1 plays critical roles in neural development, but the mechanisms by which it detects and transduces mechanical signals into cellular responses remain poorly understood. This proposal aims to elucidate how PIEZO1 mechanotransduction shapes early neural development through three specific aims: (1) Decipher the mechanistic links between cell-generated forces, membrane tension, and PIEZO1 in Neural Stem Cells (NSCs); (2) Determine the molecular mechanism underlying PIEZO1-mediated regulation of cholesterol biosynthesis in neural development; and (3) Elucidate the role of PIEZO1 in NSC and neuronal migration. The research employs innovative approaches including: quantitative imaging of endogenous PIEZO1 activity using PIEZO1-HaloTag human induced pluripotent stem cells, micropatterned substrates to control cellular mechanics, neural rosette models that recapitulate early human neural development, genetically-engineered mice, and bioengineered scaffolds that mimic radial glial tracks guiding neuronal migration. This systematic investigation spanning single molecules to developing tissue models will reveal fundamental mechanisms by which PIEZO1 coordinates cellular responses to mechanical cues during neural development. Understanding these pathways has therapeutic potential for neurodevelopmental disorders where mechanical signal transduction is disrupted.

NHLBI - National Heart Lung and Blood Institute

Phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative phenotype, plays a causal role in many human occlusive vascular diseases. To better understand key biological events occurring in human vascular diseases, we analyzed proteomic data from human atherosclerotic plaques and genomic data associated with human coronary artery disease. This unbiased analysis revealed that many genes involved in vesicle trafficking/fusion are over-represented. Previous studies have shown that the lipid kinase PIK3C3 is an essential regulator of vesicle trafficking/fusion. However, its functional role in VSMCs remains completely unknown. To examine the role of PIK3C3 in VSMCs, we generated inducible SM-specific Pik3c3 knockout (iSM KO) mice driven by Myh11-CreERT2 transgene. Unexpectedly, Pik3c3 iSM KO mice exhibited lethality 4 weeks after deletion of Pik3c3, due to a pseudo-obstructive intestine resulting from deletion of Pik3c3 in visceral SMCs in addition to VSMCs. The iSM Pik3c3 KO mice also exhibit dramatic remodeling of the vascular wall including thickening, aneurysmal dilation and spontaneous neointima. Proteomic analysis and bulk RNA- seq of Pik3c3-deficient aorta revealed loss of contractile proteins while increased expression of inflammation genes and targets of the Hippo-YAP1 pathway which has been shown to be critical for VSMC development and phenotypic modulation. Single cell RNA-seq revealed that Pik3c3-deficient aortic VSMCs almost completely lose their identity of contractile VSMCs while acquiring markers of inflammatory cells and mesenchymal stem cells. These exciting data suggest a previously undocumented role for PIK3C3 in maintaining SMC identity. Mechanistically, Pik3c3 inactivation induced YAP1 protein expression and silencing Yap1 largely restored a contractile phenotype in Pik3c3-deficient VSMCs. We hypothesize that PIK3C3 is a “master” regulator of the contractile phenotype of VSMC via regulating autophagosome-mediated degradation of YAP1. Three specific aims are proposed to test this hypothesis. To circumvent the early lethal visceral phenotype seen with Myh11- CreERT2 transgene, in Aim 1 we will employ a novel vascular-specific inducible Itga8-CreERT2 mouse to generate VSMC-specific Pik3c3 KO mice. Atherosclerosis will be induced using PCSK9 AAV and the effects of VSM- specific deletion of Pik3c3 on lesion formation will be evaluated. Wire injury-induced neointimal formation will be assessed as well by using this novel KO mouse model. Aim 2 will test that YAP1 is a critical mediator conferring the effects of Pik3c3 deficiency on VSMCs. YAP1 will be pharmacologically and genetically inactivated, and its effect on vascular remodeling and gene expression will be determined. Aim 3 will test that YAP1 protein accumulation induced by Pik3c3 deficiency is due to the impaired autophagic flux that attenuates autolysosome- mediated YAP1 degradation. Proposed studies will determine the role of PIK3C3 in autophagic flux in vivo and the role of ubiquitin and p62/SQSTM1 in PIK3C3-mediated degradation of YAP1 in human VSMCs in vitro. Completion of these studies will provide novel insights into the mechanism of controlling VSMC phenotype.

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

Child development and organogenesis depend on a highly regulated interplay of inductive events, which are regulated by evolutionarily conserved cell signaling pathways. Wnt-signaling pathways are a signaling system, conserved across the animal kingdom and regulating early development by inductive events and subsequent cell growth, cell fate, and cell polarity across whole organisms and during organogenesis in the whole body. Wnt-signaling is also critical in stem cell establishment and maintenance of (almost) all tissues. At the center of these pathways are the Wnt morphogens and their receptors, the Frizzled family (Fz in Drosophila, and Fzd in mammals) of 7-pass trans-membrane proteins. The canonical Wnt-pathway also critically employs the LRP5/6 co-receptors (known as Arrow in Drosophila). While a lot is known about the architecture of the two main Wnt-pathways: the canonical Wnt/-catenin pathway and Wnt/PCP (Planar Cell Polarity) signaling, and the membrane protein complex associated activation of the canonical pathway, employing Fzd-LRP5/6 heteromeric complexes for Wnt binding, much less is known about the activation of Wnt/PCP signaling, leading to asymmetric PCP complex localization and hence cell polarity. We have strong preliminary evidence for a critical role of Arrow-LRP5/6 in Wnt/PCP signaling, which we propose to address in this application. Regulation of Wnt-signaling specificity between the Wnt/-catenin pathway and Wnt/PCP signaling remains unclear, and has so far largely been linked to the presumed specific role of Arrow-LRP5/6 in the Wnt/-catenin pathway, and differential recruitment of Dishevelled (Dsh/Dvl) proteins as the cytoplasmic mediators of Wnt- receptor interactions. As Dsh is at the node of all Wnt-signaling events and the pathways are sensitive to levels of Dsh/Dvl, we have designed and utilized a genome-wide genetic screen in Drosophila to identify novel “pathway specificity”-regulators of Wnt-signaling activation. Strikingly, this screen has revealed a critical role of Arrow-LRP5/6 in both pathways, including Wnt-PCP signaling, while it is presumed to be Wnt/-catenin pathway specific as outlined above. We are proposing here to functionally define and dissect the role of Arrow- LRP5/6 in Wnt-PCP signaling. This new function of Arrow-LRP5/6 in Wnt/PCP signaling is very unexpected and intriguing, as it challenges the pathway architecture as presented in text books, and thus it is an exciting and challenging new research avenue in the Wnt-signaling context. The specific Aims are to (1) define the phenotypic requirements of Arrow-LRP5/6 in Wnt-PCP signaling, and (2) establish the molecular mechanisms of how Arrow-LRP5/6 intersects with the well-known PCP-Core factors, including the PCP dedicated Fz receptor subset(s) and the shared effector Dsh. Our preliminary data suggest that Arrow-LRP5/6 promotes Fz- PCP signaling, acting positively upstream of Dsh in the PCP complex formation. Information acquired in this application will advance our mechanistic understanding of Arrow-LRP5/6 in the Wnt-pathways, and potentially could also lead to follow up studies in developmental disease associated contexts.

U.S. National Science Foundation

Proposals in the area of plasma physics submitted to the Division of Physics that are not governed by another solicitation (such as CAREER), should be submitted to the Division-wide solicitation: <a href="https://new.nsf.gov/funding/opportunities/division-physics-investigator-initiated-research">Division of Physics: Investigator-Initiated Research Projects</a>. The Plasma Physics program participates in multiple NSF meta-programs such as the <a href="https://new.nsf.gov/funding/opportunities/ecosystem-leading-innovation-plasma-science" target="_blank">ECosytem for Leading Innovation in Plasma Science and Engineering (ECLIPSE)</a>, <a href="https://new.nsf.gov/funding/opportunities/windows-universe-era-multi-messenger-astrophysics" target="_blank">Windows on the Universe: The Era of Multi-Messenger Astrophysics (WoU-MMA)</a>, and <a href="https://new.nsf.gov/funding/opportunities/computational-data-enabled-science-engineering-3" target="_blank">Computational and Data-enabled Science and Engineering (CDS&amp;E)</a>. Topically appropriate proposals may also be submitted to the Plasma Physics program in response to NSF Dear Colleague Letters such as <a href="https://www.nsf.gov/pubs/2022/nsf22111/nsf22111.jsp" target="_blank">Critical Aspects of Sustainability (CAS): Innovative Solutions to Sustainable Chemistry (CAS-SC)</a>. When permitted under an MOU between NSF and another funding agency or private foundation, NSF may share information from proposals submitted to this solicitation for consideration of joint funding, and may invite employees of such organizations to attend merit review panels as observers. MOUs of relevance to the Plasma Physics program presently exist with the Department of Energy/Office of Science, National Nuclear Security Administration, the Air Force Office of Scientific Research, the US-Israel Binational Science Foundation, the Czech Science Foundation, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), and the Swiss National Science Foundation. Plasma Physics is a study of matter and physical systems whose intrinsic properties are governed by collective interactions of large ensembles of free charged particles. 99.9% of the visible Universe is thought to consist of plasmas. The underlying physics of the collective behavior in plasmas has applications to space physics and astrophysics, materials science, applied mathematics, fusion science, accelerator science, and many branches of engineering. The Plasma Physics program supports research that can be categorized by several broad, sometimes overlapping, sub-areas of the discipline, including: magnetized plasmas in the laboratory, space, and astrophysical environments; high energy density plasmas; low temperature plasmas; dusty, ultra-cold, and otherwise strongly coupled plasmas; non-neutral plasmas; and intense field-matter interaction in plasmas. The focus of the Plasma Physics program is to generate an understanding of the fundamental principles governing the physical behavior of a plasma via collective interactions of large ensembles of free charged particles, as well as to improve the basic understanding of the plasma state as needed for other areas of science and engineering. Principal Investigators (PIs) are encouraged to consider including specific efforts to increase diversity of the plasma physics community and broaden participation of under-represented groups in Science, Technology, Engineering, and Mathematics (STEM) as Broader Impacts of proposed work. Development of new undergraduate and graduate plasma physics curricula, or curricula enhancement to include plasma physics topics in other courses, at institutions lacking such coursework is similarly encouraged. NSF recognizes that some research projects within this Program may require more than three years to realize demonstrable research outcomes. For such projects, PIs are encouraged to consult the above Program Director to discuss the possibility of submitting a proposal of 4- or 5-year duration. Some Plasma Physics-related activities are supported primarily by other NSF Programs. Proposals focused on the physical properties of individual or a small number of atoms or molecules, or optical physics, should be directed to the Atomic, Molecular, and Optical Physics Program within the Division of Physics. Proposals focused on understanding astrophysical systems should be directed to the Division of Astronomical Sciences. Proposals focused on understanding the Geospace environment or the Sun-Earth interactions should be directed to an appropriate program within the Geospace Section of the Division of Atmospheric and Geospace Sciences. Proposals focused on development of new materials using plasmas should be directed to an appropriate program in the Division of Materials Research. Proposals focused on plasma-assisted manufacturing should be directed to the Division of Civil, Mechanical and Manufacturing Innovation. Finally, proposals focused on use of plasmas for environmental and reaction engineering, environmental sustainability, combustion systems, or engineering of biomedical systems should be directed to an appropriate program within the Division of Chemical, Bioengineering, Environmental and Transport systems.

Louisa Boren STEM PTA

The Louisa Boren School Community is embarking on a project to make the playground more family friendly for use by the neighborhood. The project includes the installation of a bench to serve as a resting place, a site clean up and adding paint to 4 square courts and the area adjacent to the new bench. A community celebration at the playground, 5950 Delridge Way South West, is planned for June.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) has persistently maintained a low ~32% 5-year survival rate in spite of over three decades of medical research. With approximately 22,010 in the U.S being diagnosed with AML and 11,090 dying from the disease yearly, there remains an urgent need to develop new therapies and treatment strategies. Understanding the key protein drivers of AML and the regulatory mechanisms governing expression of these proteins has provided us with an opportunity to specifically target the disease. The most common variants of AML in both pediatric and adult patients depend upon the chromatin binding MLL protein complex. Recently developed small molecule menin-inhibitors have demonstrated remarkable clinical success by inhibiting MLL complex formation. However, close to 40% of patients treated with menin inhibitors for prolonged periods develop resistance and subsequently relapse, demonstrating an urgent need for combination therapy. Our lab has previously shown that a critical component of the MLL complex, LEDGF, is sensitive to perturbations in translation due to its short half-life. I demonstrated that LEDGF protein expression can be inhibited with the RNA helicase eIF4A1 inhibitor silvestrol, and that silvestrol has potent anti -AML properties. The following aims will test the hypothesis that eIF4A1 inhibition is an effective AML therapy in vivo, capable of circumventing menin inhibitor resistance. In Aim 1 I will generate novel mouse models of menin inhibitor resistant adult and pediatric AML to test the efficacy of eIF4A1 inhibition as an AML therapy. In Aim 2 I will identify the molecular mechanisms by which eIF4A1 contributes to LEDGF translation, exploring the role of eIF4A1 in maintaining AML cells. The long-term objectives of this project are to characterize a clinically relevant new approach to treat AML and uncover molecular mechanisms governing mRNA translation. This fellowship application is sponsored by Dr. Daisuke Nakada, PhD, an expert in hematopoietic stem cell biology and acute myeloid leukemia biology. This training plan is designed to 1) provide mentorship from experts in science and medicine; 2) develop general and field-specific scientific knowledge in stem cell, chromatin, and RNA biology; 3) grow my scientific communication skills and form professional networks; and 4) develop clinical skills and knowledge toward a pediatrician-scientist career. The clinical and scientific training environment is at Baylor College of Medicine, located in the heart of the Texas Medical Center with close ties to institutions such as Texas Children's Hospital and MD Anderson Cancer Center. This environment is ideal to foster scientific and clinical growth toward my long-term goal of becoming a physician scientist in the field of pediatric hematology-oncology.

NIGMS - National Institute of General Medical Sciences

Project Summary P-bodies are ribonucleoprotein granules that form membraneless compartments through liquid-liquid phase separation and regulate gene expression by directing mRNAs for degradation, translation, or storage. In humans, P-bodies are estimated to influence one-third of genes in the genome. Recent research highlights their involvement in stem cells, where they regulate cell cycling and cell fate decisions. Despite these advances, little is known about how P-bodies function in connective tissue or during tissue regeneration. Our recent work using in vivo proximity labeling in zebrafish identified Ddx61, a protein central to P-body formation, as critical during regeneration. Ddx61 forms condensates reminiscent of P-bodies, and its loss is associated with reduced cell proliferation and impaired tissue regeneration. Interestingly, these regenerative P-bodies form in response to injury and dissipate once regeneration is complete, suggesting they are dynamically regulated. These findings provide a strong foundation to explore the mechanisms of P-body formation and their functional significance in regeneration. In my newly established laboratory, we aim to address the mechanisms underlying P-body formation and function in the context of tissue regeneration. Specifically, we will determine the composition of regenerative P-bodies using innovative tools, including in vivo proximity labeling and super-resolution microscopy. To establish causality, we will bioengineer artificial P-bodies and test their ability to regulate mRNA fate in zebrafish. This will be one of the first comprehensive studies on the post-transcriptional regulation of gene expression by P-bodies in connective tissue and during tissue regeneration. By uncovering how regenerative P-bodies regulate cell division and differentiation, this research will advance our understanding of post-transcriptional regulation and provide a foundation for developing novel therapeutic strategies for tissue regeneration, aging, and cancer.

European Commission - HORIZON

Pragmatic clinical trials to optimise immunotherapeutic interventions for patients with refractory cancers. Call: Supporting the implementation of the Cancer Mission. Programme: HORIZON. Keywords: Anticancer therapy, Diagnostic tools (e.g. genetic, imaging), Haematology, Medical devices, Paediatrics, Pharmacogenomics, Pharmacology, pharmacogenomics, drug discovery and design, drug therapy, Radiation therapy, Stem cell therapy, Surgery. Open call from the EU Funding & Tenders Portal.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

TITLE: PRAISE (Pressure Relief Assessment Information System): A Paradigm-shifting Mobile Health Platform for Pressure Relief Adherence in Manual Wheelchair Users PROJECT SUMMARY: The proposed project aims to create a pressure relief assessment information system (PRAISE) to enhance the adherence of manual wheelchair users to Clinical Practice Guidelines (CPGs) designed to prevent pressure ulcers. The motivation of this research stems from two core challenges. First, pressure ulcers pose a serious threat to manual wheelchair users with spinal cord injuries, frequently leading to painful complications, infections, and even premature death. To reduce pressure ulcer risks, CPGs recommend that wheelchair users perform pressure relief activities (i.e., vertical pushups, lateral, and forward leans) every 15 to 30 minutes. However, research reveals that wheelchair users may not adhere to CPGs in everyday life. Second, no universally adopted tools currently exist to monitor CPG adherence, nor is the understanding of factors leading to non-adherence. As a result, the prevalence of pressure ulcers among wheelchair users with spinal cord injuries remains high. Built upon the International Classification of Functioning, Disability and Health (ICF) model, PRAISE will shift from the conventional singular focus on adherence to a holistic approach, which will cohesively integrate a user's health, personal, and environmental factors through its multidimensional design. First, PRAISE will enable users to create profiles, including demographics, wheelchair usage patterns, and medical records related to pressure ulcers. Second, this foundational data will be augmented by a spectrum of sensor data (i.e., accelerometer, heart rate, GPS, and battery life) from a smartwatch, critical for ecological momentary assessments (EMAs). Third, our novel distributed algorithm can accurately detect pressure relief activities without relying on frequent, costly internet connections. It achieves this through lightweight processing on mobile devices to capture patterns intrinsic to pressure relief activities, hence transmitting only relevant data segments to the server for fine-grained recognition. Fourth, grounded in the ICF framework, PRAISE will dynamically integrate user-specific health, personal, and environmental factors to deliver context-aware feedback and personalized guidance. Through reinforcement learning, PRAISE will continuously evolve its guidance by learning from user responses and behavior, ensuring that interventions remain effective and tailored to individual needs over time. In collaboration with a diverse advisory team, PRAISE's development will prioritize robust security, user- friendliness, advanced analytics, and customizable assessment modules. Once the advisory team completes the initial validation, a feasibility and acceptability assessment will be conducted by involving 15 manual wheelchair users for two weeks. To gain a deeper understanding of user experiences, we will employ multifaceted approaches to gather and analyze user feedback. As PRAISE strives to make pressure ulcer prevention more accessible and personalized for wheelchair users, it will help reduce health disparities, particularly for those who may not have easy access to traditional healthcare resources. Therefore, PRAISE will revolutionize care for the manual wheelchair users to achieve patient-centric, evidence-based interventions.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT A lack of proven interventions to prevent leukemia in aged populations leaves a growing demographic vulnerable to this devastating disease. While leukemic cells are subject to immune selection which influences disease progression, we lack knowledge of the stages at which T cells shape the hematopoietic stem and progenitor cell (HSPC) pool from the initiation of clonal hematopoiesis (CH) through to the progression to leukemia, which limits our ability to intervene in this process. The long-term goal of this project is to identify immuno-preventative strategies to intercept leukemogenesis at its earliest stages. The overall objective of this application is to determine the mechanisms by which, and at which stages of pre- leukemic development, HSPC clones are detected and selected by the adaptive immune system. The central hypothesis is that reduced IFNγ responsiveness enables immune evasion of CH-mutant (Dnmt3amut) HSPCs thereby promoting clonal expansion and pre-leukemic evolution. The rationale is grounded in the observation that HSPCs from humans and mice with recurrent CH driver mutations in Dnmt3a have reduced transcript and protein expression of MHC-II machinery, and reduced presentation of exogenous and endogenous antigens via MHC-II. Mechanistically, MHC-II is potently induced by IFNγ on wild-type HSPCs but to a lesser extent on Dnmt3amut HSPCs. In vitro and in vivo, we observe less activation and proliferation of CD4+ T cells by Dnmt3amut HSPCs compared to control HSPCs, supporting that Dnmt3amut HSPCs have reduced immunogenicity. The central hypothesis will be tested by pursuing two specific aims: 1) to define the stages of pre-leukemic HSPC selection that are controlled by CD4+ T cells, and 2) to evaluate decreased IFNγ response of pre-leukemic Dnmt3amut HSPCs as a mechanism of immune evasion. This research is innovative because it introduces a novel framework for understanding how adaptive immunity shapes clonal evolution of pre-leukemic HSPCs. While considerable attention has been given to genetic and cell-intrinsic drivers of CH, the role of immune surveillance—particularly adaptive immune selection—in governing HSPC clonality remains largely unexplored. Ultimately, the proposed work is significant because it will define the role of CD4⁺ T cells in HSPC clone selection during early disease phases in CH and pre-leukemia which has major therapeutic implications for immunoprevention of leukemia.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract The ultimate success of immunotherapy for brain malignancies, such as malignant glioma, will require integration of in-depth understanding of immunology with solutions for the following long-standing challenges: 1) paucity and heterogeneous expression of glioma-specific antigens; 2) on-target off-tumor toxicity and exhaustion of therapeutic T lymphocytes, such as chimeric antigen receptor (CAR) T-cells; 3) immunological privilege of the CNS and 4) immunosuppression involving tumor, neuronal, and immune cells. My laboratory has contributed to critical discoveries in these areas and integrated our findings into novel immunotherapy clinical trials for glioma patients. In the current proposal, I will enhance my research by mobilizing multiple immune mechanisms. To this end, I will collaborate with an outstanding group of investigators whose diverse expertise in multi-disciplinary areas complements my own in brain tumor immunology as the central component and apply a wide variety of resources available at UCSF and collaborators to one overarching program. I will evaluate the overarching hypothesis that the integration of novel cell-engineering and antigen-targeting approaches will allow us to develop safer and more effective immunotherapy strategies by overcoming heterogeneous expression of antigens and unique challenges in brain immunology. I will evaluate the following strategies: 1. Develop neo- junction-targeting T-cell receptor (TCR)-T cell-based immunotherapy. We will leverage our highly reliable and valuable pipeline for T-cell epitope prediction, which we established during the current funding cycle, to discover novel neoepitopes derived from tumor-specific alternative splicing events (neojunctions). 2. Develop novel cell therapies using allogeneic induced pluripotent stem cells (iPSCs) and in vivo transduction approaches. While my current NINDS R35 award allowed me to implement the first-in-human phase I study of Synthetic Notch (synNotch)-CAR T-cell therapy in patients with glioblastoma, inherent and logistical challenges associated with the use of autologous T-cells motivate us to develop these novel and alternative approaches. 3. Enhance “epitope spreading” to overcome the antigen heterogeneity. While the novel synNotch-CAR approaches are promising, one major inherent challenge is that targeting a few or several antigens by CARs or TCRs may not adequately cover the marked antigenic heterogeneity of tumors. We will enhance the effects of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce adaptive immune responses against heterogeneous tumor antigens. 4. Investigate the glioma-neuronal circuit-induced immune regulation. We will delineate essential mechanisms on our recent discovery of neuronal activity-driven immunosuppression as a previously unrecognized resistance mechanism of cancer immunotherapy for gliomas. These 4 strategies will be logically integrated into combination approaches. As expected per the purpose of the NINDS R35 mechanism, these strategies may involve high risks. However, based on our preliminary proof-of-principle data, we will persistently pursue our goals with long-term support from the R35 mechanism and adopt new technologies flexibly and swiftly.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Patients with obstructive vascular diseases, such as atherosclerosis or peripheral arterial disease, or acute peripheral injuries require vascular grafts to restore blood flow to areas of the body. While the use of autologous vessels is considered the gold standard of treatment, many patients lack suitable vessels due to either vascular disease, previous usage, or have a size mismatch to the injured vessel. Therefore, clinicians turn towards synthetic grafts, such as expanded polytetrafluoroethylene (ePTFE) or Dacron, for large diameter vessel reconstruction. However, these synthetic materials fail when used in clinical small-diameter vascular applications, requiring the development of novel, hemocompatible vascular grafts for these clinical needs. Previous clinical trials have investigated acellular tissue-engineered vascular grafts (TEVGs) developed using human primary smooth muscle cells seeded on biodegradable scaffolds. After robust extracellular matrix (ECM) deposition, these TEVGs were subsequently decellularized and directly investigated for vascular treatment. While promising, the acellular TEVGs lacked an endothelium, and resulted in significant occlusion and suboptimal function within patients. Therefore, developing a novel TEVG with a functional endothelium that is immunocompatible to any recipient is of great clinical need. To address this issue, we propose using human induced pluripotent stem cells (hiPSCs) to fabricate a robust TEVG lined with an endothelium that is universally accepted by any patient, mitigating allogeneic immunorejection. In this proposal, hiPSCs will be differentiated into vascular smooth muscle cells (VSMCs) and subsequently used to generate a robust TEVG in our bioreactors that is then decellularized. Of novelty, we will then endothelialize the TEVGs with hypoimmunogenic, “universal” endothelial cells (ECs) that have been previously developed in our lab by modulating human leukocyte antigens (HLA) expression. To avoid xenograft immunorejection, this proposal will develop and characterize universal pig iPSCs (piPSCs) to endothelialize the TEVGs through downregulating expression of MHC I and II molecules and upregulating expression of CD47 via CRISPR-Cas9. The aims of this grant are to (1) characterize hypoimmunogenic universal piPSC lines for vascular graft engineering and (2) to generate universal iPSC- TEVGs and investigate their hemocompatibility in a preclinical porcine carotid bypass model in vivo. By investigating the universal iPSC technology in a preclinical porcine model, future studies will investigate human universal iPSCs for vascular tissue engineering purposes, furthering our goal towards developing a universal vascular conduit accepted by any patient.

NIGMS - National Institute of General Medical Sciences

Convergent molecular evolution, especially among distantly related species, is a hallmark of adaptation, yet the drivers of such convergence (or lack thereof) are typically unknown. Variation in molecular convergence may stem from constraints on evolutionary trajectories, such as how intramolecular epistasis and broader scale interactions among genes differ across lineages. While substantial progress has been made in understanding the prevalence of epistasis for fitness-related phenotypes, particularly in microbial systems, empirical tests of the role of epistasis in convergent molecular evolution are rare, especially in metazoans. A key obstacle is the lack of tractable, highly replicated systems to investigate the extent and generality in the causes of molecular convergence. To meet this need, we have been studying a diverse group of insects which have adapted to cardenolides, a class of steroidal plant toxins that disrupts the biomedically-relevant animal protein, Na/K-ATPase. We recently documented a remarkable 30 independent origins of cardenolide-specialization in insects, spanning 350 million years of evolution (in six taxonomic orders, spanning beetles and flies to grasshoppers). Although a handful of substitutions did indeed convergently evolve in all orders, some species lack these substitutions and others have taken alternative paths. Our findings, which also show distinct patterns among groups (e.g., Coleoptera vs. Lepidoptera, each with multiple origins) suggests lineage-specific constraints of genomic background. This group of insects thus presents a treasure trove of opportunity to decipher the drivers of molecular convergence. How variable are the epistatic interactions between lineages, and do these differences drive alternative outcomes in molecular evolution? Do multiple genes coevolve, shaping patterns of convergence? For example, have ABC transporter genes involved in excretion and storage, which complement resistance to cardenolides, evolved in parallel to Na/K-ATPase substitutions? And finally, do molecular substitutions predictably track the evolution of specific toxins coevolving in host plants? This system allows for some of the strongest general tests of why adaptive phenotypic outcomes have a similar genetic basis. Beyond comparative genomics, which will reveal distinct evolutionary outcomes and genetic associations, we will integrate the power of transcriptomics, in silico models, and functional assays to directly test our hypotheses. Our five-year program is expected to reveal general rules governing when intramolecular epistasis versus broader interactions among genes drive molecular convergence.

NIA - National Institute on Aging

PROJECT SUMMARY The mission of our laboratory is to pursue answers to essential questions in the field vascular aging that will advance our basic understanding and translate into more effective treatments to optimize human vascular healthspan. The central thesis of this project is that endothelial cells differentiated from hiPSCs, obtained from a diverse group of healthy adults and those with vascular contributions to cognitive impairment and dementia (VCID), can be leveraged to study endothelial aging in dementia. Using a computational model to identify biosignatures that predict endothelial cell aging, we will leverage this information to probe mechanisms relevant to dementia. Our research bridges the fields of vascular biology, stem cell biology, epigenetic clocks, multi -omics, and computational modeling to close the gap in the availability of models for the study of endothelial aging in dementia. There is a tremendous opportunity to address outstanding questions in this field using the novel human induced PlurIPotent stem cell-endothELIal cell model of aging for the study of vascular coNtributIoNs to coGnitive impairment and dementia (PIPELINING) described in this application. We will (1) passage human induced pluripotent stem cells differentiated to endothelial cells (hiPSC-ECs) and identify aging endpoints modeled in vitro (mitochondrial function, senescence, and angiogenesis). (2) A computational multi-scale model will be developed to predict the aging endpoints using multi -omic biosignatures for each human donor and passage. (3) Biosignature covariates judged to be critical contributors to the PIPELINING model will be selected for further mechanistic study. Achievement of the PIPELINING model would represent a significant advance in the application of contemporary technologies (iPSCs, epigenetic clocks, -omics, computational multi-scale modeling) to VCID.

NIMH - National Institute of Mental Health

Project Summary Fragile X syndrome (FXS) stands as a prominent contributor to intellectual disability and autism spectrum disorders, stemming from mutations within the FMR1 gene. These mutations lead to severe reduction or absence of the FMRP protein. Despite extensive research, effective medical interventions for FXS remain elusive, hindered by a limited understanding of its underlying mechanisms. Biochemical investigations have consistently highlighted FMRP's role in modulating mRNA translation, with its absence correlating with increased translation levels of select FMRP- interacting mRNA targets. However, emerging evidence suggests broader dysregulation, as FXS neurons exhibit heightened overall protein synthesis, hinting at elevated translation of non-FMRP interacting mRNAs. This intriguing phenomenon underscores the need for a deeper exploration into the cellular dysfunctions characterizing FXS. This research initiative aims to unravel a novel facet of FXS pathology—nucleolar hyper-function. We propose that this hyper-function contributes to aberrant ribosome biogenesis, thus augmenting the cellular capacity for translation and driving the observed global increase in protein synthesis in FXS. Aim 1 will assess neuronal and glial nucleolar function in wild-type (WT) and Fmr1 knockout (KO) mice. Aim 2 will conduct a comparative analysis of genome-wide proteomic data encompassing nucleolar proteins in WT and Fmr1 KO samples, discerning molecular alterations integral to ribosome biogenesis and assembly. Aim 3 will assess nucleolar function in the peripheral tissue in Fmr1 KO mice, establishing the hyper-functional pathological outcome as a potential clinical biomarker. The successful execution of this exploratory R21 project promises to unveil previously unexplored cellular mechanisms underlying FXS pathology. This study will also suggest nucleolus-associated abnormalities as novel molecular/cellular measures and potential biomarkers.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary In the human brain, a large repertoire of ion channels regulates the excitability of neurons, circuits, and networks, generating complex human cognition and behavior. Most ion channels are encoded by long, multi- exon genes which undergo extensive alternative splicing. Dysregulated splicing can alter ion channel function and is implicated in disorders ranging from autism to epileptic encephalopathy. Specific splicing events in ion channels have been shown to be critical for brain development and homeostasis, such as the neonatal-to-adult splicing switch in voltage-gated sodium channels. Splice-modulating therapeutics, such as antisense oligonucleotides, have shown efficacy for multiple neurologic disorders including spinal muscular atrophy, Duchenne muscular dystrophy, and Dravet syndrome. As potential targets for both small molecules and RNA therapeutics, ion channels are particularly important candidates for the treatment of neurologic disorders. However, there has never been a systematic study of alternative splicing of ion channels to-date. This proposal applies two innovative methods to profile the ion channel transcriptome and study the functional impact of alternative splicing on neuronal physiology. Aim 1 combines long-read RNA-sequencing and transcript capture technology to comprehensively identify and annotate channel isoforms in the human cerebral cortex. Bioinformatic tools will be used to uncover ion channel isoforms which are differentially regulated during postnatal brain development. Aim 2 employs splice modulation technology to study the functional impact of alternative splicing on neuronal physiology, using the epilepsy-associated KCNMA1 gene as proof of concept. A Cas-based toolkit is developed for human stem cell-derived neurons to manipulate the splicing of KCNMA1, and whole-cell patch-clamp physiology measures the impact of splice modulation on neuronal excitability. Taken together, this proposal will generate the most comprehensive profile of ion channel isoforms to-date, uncover developmentally regulated splicing events that can be potentially targeted by RNA therapeutics, and demonstrate proof of concept for transcriptome engineering in human neurons. These skills and resources will catalyze my career as an independent researcher, which will focus on the study of alternative splicing and the development of splice-modulating therapeutics for epilepsy and other neurologic disorders.

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

PROJECT SUMMARY: The mechanisms by which stem cells orchestrate their program to become functional differentiated cells require accurate temporal regulation of specific gene expression programs. This complex network requires precise temporal regulation of transcription and degradation processes to activate specific programs in a coordinated manner. So far, most of the studies have explored the regulation of transcriptional pathways and chromatin remodeling events during the differentiation process. mRNA degradation processes may present an attractive and still poorly explored opportunity for enhancing our understanding of the differentiation process. However, the lack of technologies that can capture rapid mRNA degradation events over highly dynamic processes, such as differentiation, and the heterogeneity of the mRNA degradation machinery in composition and expression patterns during differentiation have presented major technical limitations to further exploring the role of mRNA degradation across the continuum of the differentiation program. Here I propose to explore the existence of specialized RNA degradation complexes that control the decay of specific mRNA subclasses at precise timeframes of the differentiation process. To test this, we will introduce a new platform that uses cutting-edge technologies integrated in an innovative way to interrogate the continuum of the differentiation process at an unprecedented resolution. Our programmable depletion and rescue strategy will allow us to control the expression level of each subunit of complex mRNA degradation machinery robustly and with a precise time resolution of hours. By combining this technology with a high-content imaging system, we can record phenotype changes and accurately determine the specific impact of any perturbed protein on differentiation. Additionally, the use of this platform will guide us to understand the exact gene regulatory network controlled by the machinery at the transcriptional and stability level. The conceptualization and development of this workflow have the potential to impact a broader scientific audience; due to its extremely high flexibility, it could be applied to the study of unlimited biological processes or proteins. In this essay, the application of our proposed platform has the potential to fundamentally overturn the current view of how mRNA decay is dynamically regulated, providing a definite understanding of the function of the degradation machinery on mRNAs and, at the same time, revealing the broader impact of the degradation process on differentiation.

U.S. National Science Foundation

The Promoting Research and Innovation in Methodologies for Evaluation (PRIME) program seeks to support research on evaluation with special emphasis on exploring innovative approaches for determining the impacts and usefulness of STEM education projects and programs; building on and expanding the theoretical foundations for evaluating STEM education and workforce development initiatives, including translating and adapting approaches from other fields; and growing the capacity and infrastructure of the evaluation field. Two types of proposals will be supported by the program: Exploratory Projects that include proof-of-concept and feasibility studies and more extensive Full-Scale Projects.

NCI - National Cancer Institute

Project Summary The goal of this project is to develop a strategy to effectively eradicate leukemia-initiating cells. Leukemia- initiating cells are responsible for tumor initiation and recurrence in acute myeloid leukemia (AML), making it critically important to understand and target the biology required for leukemia-initiating cell survival. LICs are characterized by their self-renewal capacity, block in differentiation, and quiescent nature making them therapy resistant. A well characterized vulnerability of leukemia-initiating cells is oxidative phosphorylation (OxPhos) a pathway responsible for energy production. Direct OxPhos inhibition has been toxic in cancer patients. Thus, the development of approaches to target processes that regulate OxPhos in leukemia-initiating cells that are dispensable in normal cells is required. Our preliminary data shows that OxPhos is regulated by a post- translational modification called protein glutathionylation in AML cells and leukemia-initiating cells but not in normal hematopoietic stem and progenitor cells (HSPCs). These data indicate that protein glutathionylation regulation may represent a mechanism for decreasing OxPhos that could be LIC/AML specific and therefore targeting protein glutathionylation may be an approach to kill LICs with a more favorable therapeutic window than other approaches. Importantly, our data suggests that depletion of mitochondrial proteins that regulate protein glutathionylation results in reduced LIC function, induction of myeloid cell differentiation and sensitizes primary human AML cells to commonly used AML therapies but does not impact HSPCs. These data further support the potential for a therapeutic window may exist to target protein glutathionylation in AML. Based on these findings, we hypothesize that the regulation of mitochondrial protein glutathionylation is essential for LIC function by regulating OxPhos. We will examine this hypothesis by determining the molecular and biological role of protein glutathionylation in regulating leukemia-initiating cells and HSPC function using primary AML specimens, patient derived xenograft (PDX) models, and normal bone marrow specimens from healthy donors. Specifically, we will quantify leukemia-initiating cell phenotypes and function upon genetic depletion of proteins that regulate glutathionylation. Further, we will interrogate the mechanism(s) by which protein glutathionylation regulates mitochondrial energy production in leukemia-initiating cells. Taken together, our studies will be the first to establish protein glutathionylation as a novel regulator of 1) leukemia-initiating cells function and 2) OxPhos in cancer.