Massachusetts Institute Of Technology

NIH Research Projects · FY 2025 · 2022-09

Astrocytes are the major non-neuronal cell type in the cortex and are increasingly recognized as key contributors to the development, plasticity and function of neuronal circuits. Yet, how they participate with neurons in learned behavior and dynamically shape the underlying cortical circuits is poorly understood. The primary motor cortex is required for learning and executing voluntary movements: the acquisition of a cued, stereotyped, movement in mice is accompanied by synaptic remodeling of motor cortex neurons and the emergence of coordinated movement-related ensemble neuronal activity. Here, we propose to examine functional astrocyte mechanisms in motor cortex that mediate synaptic plasticity and neuronal dynamics during motor learning. Astrocytes have highly ramified fine processes that contact nearly all synapses in the cortex, where they modulate synaptic transmission and plasticity by mechanisms that include uptake of glutamate and GABA, primarily via the transporters GLT1 and GAT3 respectively. Astrocytes also respond to, as well as modulate, synaptic activity with spatiotemporally heterogeneous calcium transients in their processes, termed microdomains. We will examine the role of astrocytes in shaping motor cortex circuits as mice learn a forelimb lever push movement, including cued response onset and reliable movement trajectory, using a range of cutting-edge approaches: simultaneous high-resolution imaging of astrocytes and neurons in vivo, computational encoding-decoding models of astrocyte and neuronal activity, astrocyte-specific gene expression analyses, and novel astrocyte optogenetic and CRISPR tools alongside established chemogenetic and viral knockdown methods. Building on our preliminary data, which demonstrate parallel learning-related changes in astrocyte microdomain responses and neuronal responses, along with gene expression changes in astrocyte GLT1 and GAT3, in Aim 1 we will determine functional astrocyte calcium signatures in motor cortex during learning and their relationship to neuronal activity and behavior. We hypothesize that astrocytes shape neuronal plasticity during task learning with corresponding plasticity in their microdomain calcium responses, which we will specify computationally. In Aim 2, we will determine the effect of astrocyte calcium signaling on motor learning and neuronal responses. We hypothesize that disruption of calcium transients alters the emergence of neuronal ensembles and expert behavior, potentially by altering astrocyte gene expression of transporter mechanisms. In Aim 3, we will determine the role of astrocyte neurotransmitter transporter function in motor cortex circuits and learning. We hypothesize that disrupting astrocytic modulation of excitatory transmission via GLT1, and inhibitory neurotransmission via GAT3, disrupts astrocytic calcium responses together with neuronal circuit plasticity and behavior. Together, these studies will provide a mechanistic, computational view of astrocyte involvement in the function and plasticity of cortical circuits, reveal their task-specific contributions to neuronal responses and learned behavior, and provide the basis for understanding their role in a range of brain disorders and diseases.

NIH Research Projects · FY 2025 · 2022-09

Summary Over the past three decades, the Jacks laboratory has been a recognized leader in the development and characterization of genetically engineered mouse models of cancer, among other pre-clinical models. The laboratory has also studied human cancer specimens and datasets to validate finding from their experimental systems and to advance discoveries toward clinical translation. While Jacks laboratory has investigated many cancer types over time, this proposal is focused on models of lung adenocarcinoma and pancreatic ductal adenocarcinoma. By developing and deploying tools of genetic engineering and genetic profiling, such as CRISPR-based methods and single-cell analysis, the laboratory has pioneered new models and analytical approaches that have allowed for a deeper understanding of disease progression, including interactions between developing tumors and the immune system. This proposal builds on this foundation at the intersection of cancer biology and technology development to explore in detail the molecular and cellular aspects of tumor evolution. Single-cell profile methods will be augmented by spatial transcriptomics to characterize the changes in gene expression—in cancer cells as well as other cell types within the tumor microenvironment—in situ, rather than in dissociated cells. Genes and pathways implicated by this analysis will be subjected to functional analysis using organoid-based models as well as in the autochthonous setting. A second major theme of this proposal is the further exploration of tumor-immune interactions in lung cancer, which the laboratory has been studying for several years. Following up on experiments investigating the factors that control T cell activation and dysfunction in the setting of lung and pancreas cancer development, the laboratory will explore methods to provoke effective anti-tumor T cell responses as well as an improved response to immunotherapy. These studies will investigate the nature of the antigens and antigen combinations that induce effective T cell priming and activation, including through prophylactic and therapeutic vaccinations. Results of these experiments will inform new therapeutic approaches, including novel cancer vaccine strategies, in human cancer patients.

NIH Research Projects · FY 2025 · 2022-09

Project Summary DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. These events begin during G1, when two copies of the replicative helicase, the Mcm2-7 complex, are loaded at all potential origins of DNA replication. Once loaded, the two ring-shaped, heterohexameric Mcm2-7 complexes encircle the DNA and interact tightly via their N-terminal domains. Although inactive, the resulting head-to-head Mcm2-7 double hexamer licenses each origin for subsequent bidirectional initiation upon entry into S phase. Consistent with their importance, mutations in or misregulation of the proteins mediating helicase loading lead to cancer and developmental abnormalities. Thus, understanding the mechanism of these processes will provide critical information concerning the maintenance of genome integrity and potential targets for therapeutics. The biochemical reconstitution of helicase loading using budding yeast proteins has been a powerful tool to understand these events, however, bulk biochemical assays are poorly suited to study the complex dynamics involved in helicase loading due to their frequently incomplete and asynchronous nature. Single- molecule fluorescence microscopy experiments bypass these problems by monitoring events on individual DNA molecules in real time, defining the sequence of biochemical events, detecting short-lived intermediates and specific protein-protein interactions, and defining quantitative kinetic mechanisms. We propose single- molecule experiments on helicase loading using reconstituted yeast proteins in vitro, supplemented with molecular genetics experiments on live cells. Together, these studies will provide critical insights into the dynamic mechanisms of helicase loading and will complement and aid in the interpretation of the static structures revealed in recent cryoelectron microscopy studies. The proposed research primarily focuses on events of helicase loading that are conserved across all eukaryotic organisms. Both yeast and metazoan ORC induce a strong bend in the DNA upon binding. In Specific Aim one, we investigate the role of this activity in origin selection and determine which steps in helicase loading require this function. The MO complex is a key helicase-loading intermediate that ensures the second recruited Mcm2-7 forms head-to-head interactions with the first. In Aim two, we will determine the role of this complex in closing of the Mcm2-7 ring around DNA and define the pathways by which ORC and Mcm2- 7 form this complex. In the final Aim, we will determine how nucleosomes and sequence-nonspecific ORC DNA binding change helicase loading, both key elements of origin selection in metazoan species.

NIH Research Projects · FY 2025 · 2022-09

Exceedingly high mutation rates permit most RNA viruses to rapidly explore protein sequence space. On the other hand, high mutation rates also result in widespread production of viral protein variants with poor biophysical properties and severe folding defects. Protein variants that cannot fold successfully are removed from the population, even if they could otherwise confer a beneficial adaptive function. Recent work has revealed that the composition and activities of the host cell’s protein folding and quality control machinery (the proteostasis network) play a central role in defining the amino acid sequence space accessible to rapidly evolving RNA viral proteins. This phenomenon has so far largely been explored using proteostasis modulation itself as the selection pressure. It is not yet clear whether host cell chaperones are directly – by enhancing viral protein folding – impacting the ability of viruses to adapt to and escape from external selection pressures stemming from the host’s adaptive immune system, antiviral drugs, or other factors. Using influenza as a model system, this proposal integrates state-of-the-art chemical biology, genetic, biochemical, biophysical, and computational methods to comprehensively evaluate and elucidate, at the molecular-level, the emerging and complex interplay between host proteostasis and viral adaptation in the context of diverse selection pressures. Aim 1 focuses on the mechanism by which hijacked host chaperones promote influenza escape from innate immune system factors, establishing biophysical origins of host chaperone-dependence in influenza nucleoprotein evolution and elucidating whether and how the virus can readily adapt to challenging host proteo- stasis environments. Aim 2 establishes how the composition and activities of the host cell’s endoplasmic reticulum proteostasis network impact the ability of influenza hemagglutinin, the primary target of influenza-neutralizing antibodies, to escape selection pressure from the adaptive immune system. Aim 3 operates on a broader scale to understand how host proteostasis networks impact genome-wide mutational tolerance and influenza error catastrophe, a phenomenon in which increasing viral mutation rates past a certain threshold causes population extinction. Experimental findings from all these Aims are integrated with protein biophysical studies and computational modeling to illuminate molecular origins of host proteostasis-dependent viral adaptation. This work is expected to establish host proteostasis as a defining force that shapes viral adaptation, particularly in the context of highly relevant selection pressures. Beyond fundamental elucidation of viral evolution, findings will greatly enhance understanding of the factors involved in viral adaptation to host selection pressures and, in the longer-term, improve the ability to accurately predict viral evolution. Discoveries are also expected to highlight the potential of therapeutic adjuvants targeting host chaperones to enable treatment regimens to which viruses cannot easily evolve resistance. Contributions will impact fields ranging from basic virology and vaccine and antiviral drug development to evolutionary biology and protein folding biophysics.

NIH Research Projects · FY 2025 · 2022-09

Summary Alzheimer's disease (AD) is pervasive and debilitating, with no truly effective treatments. Genome wide association studies have found risk variants for sporadic, late-onset AD, but the mechanisms driving this risk are still unknown. Two of the sAD variants with the highest association with development of AD are in Apolipoprotein E (APOE) and ATP-binding cassette transporter A7 (ABCA7), both of which are involved in lipid metabolism. Our prior work demonstrates that the E4 allele of APOE (APOE4) has cell type specific effects, including alterations in lipid metabolism, but important questions remain about the downstream pathways affected by this allele. Critically, we do not know how APOE4-induced changes interact with aging-related stress, leading to late-onset disease. Even less is known about how ABCA7 alleles lead to increased risk of AD. We propose to use a systems biology approach to discover these AD-risk pathways, responding to NOT-AG-18-052 from the NIH, which designates “systems biology of brain neural cells derived from human AD induced pluripotent stem cells” as a high-priority research topic. Our approach uses multi-omic analysis of induced pluripotent stem cell (iPSC) lines that are isogenic for two risk variants, APOE4 or ABCA7 premature termination (PTC), which can then be differentiated into diverse cell types. Using an unbiased approach, we will reveal how AD-risk alleles alter signaling, metabolism, and states of the cells, how they affect individual cells as well as cell-cell interactions in complex cultures, and how they alter cellular responses to acute stress. In Aim 1 we will deeply characterize the effects of APOE4 and ABCA7 PTC in 2D culture models of neurons, astrocytes, microglia and pericytes, differentiated from isogenic iPSC lines, examining changes in metabolism and post-translational modifications (PTMs) of proteins. We will use advanced network optimization methods to integrate the disparate data and to uncover molecular interaction networks that link together changes observed in the individual omics. In Aim 2, we investigate the pathways altered by risk alleles that influence cell-cell interactions in 3D culture models, using spatially-resolved PTM- proteomics and metabolomics/lipidomics and causal computational models. In Aim 3, we will examine the intersection of risk variant with environmental and cellular stressors in the 3D culture models. Each aim includes rigorous testing of hypotheses in vitro and by examination of postmortem samples.

NIH Research Projects · FY 2025 · 2022-09

Abstract Alzheimer’s disease (AD) and AD-related dementias (ADRDs) are major drivers of mortality, morbidity, and health care costs for patients and their loved ones, due to the aging population, lack of predictive diagnosis, and lack of effective treatments or prevention. Vascular contributions to cognitive impairment and dementia (VCIDs) are key contributors to AD and ADRDs and manifest through diverse cerebrovascular lesions, including atherosclerosis, microinfarcts, and small vessel strokes. VCIDs include cerebral small vessel disease (CSVD), cerebral amyloid angiopathy (CAA), and a monogenic familial form of CSVD (CADASIL, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). Understanding the mechanisms and drivers of VCID will enable new biomarkers and therapeutics, similar to the success of addressing cardiovascular disease and hypertension in heart disease. To understand the cellular mechanisms underlying VCID across brain regions, cell types, pathology, and molecular pathways, we perform high- resolution profiling of epigenomic and transcriptional alterations in post-mortem CNS samples from both sporadic and genetic VCID patients. Aim 1: We profile single-nucleus RNA-sequencing (snRNA-seq) and DNA accessibility (snATAC-seq) to create a transcriptional and epigenomic atlas of VCID across diagnoses, brain regions, cell types, sexes, and individuals. Aim 2: We create an atlas of SVD-associated changes in genes, modules, pathways, and cell-cell interactions. Aim 3: We predict candidate driver genes, regulators, and pathways using causality analyses across temporal and genetic models, and we validate our results experimentally using imaging studies. The successful execution of our studies will delineate clinically-relevant VCID biomarkers and therapeutics across sporadic and genetic VCID, enabling us to dissect their common and distinct molecular circuits, across four affected CNS region and all major cell types within them, and capturing an unprecedented level of complexity and enabling rich computational comparisons. The datasets generated and the computational analyses will provide invaluable insights for addressing the pressing medical need of VCIDs, their temporal, region-specific, and cell-type-specific changes, which can help guide new therapeutics.

NIH Research Projects · FY 2025 · 2022-09

Project summary Cells exist in a physical world, and there is often a physical basis for human function and disease. Mechanotransduction is the molecular process by which cells sense and respond to mechanical signals in their environment. Abnormal mechanotransduction can contribute to many human diseases including asthma, heart failure, osteoporosis, and cancer. Thus, it is crucial to understand the molecular basis of mechanotransduction and how these signaling pathways are disrupted during disease. Integrin receptors are critical regulators of mechanotransduction at the plasma membrane that signal through the assembly supramolecular complexes termed “focal adhesions.” Focal adhesions physically connect the actin cytoskeleton to the extracellular environment, and forces generated in the actin cytoskeleton are transmitted across focal adhesions to drive tissue morphogenesis, cell movement, and extracellular matrix remodeling. Although the proper regulation of focal adhesions is essential for integrin-dependent mechanotransduction, important questions about their formation and function remain unanswered. We do not understand how focal adhesions form, how they grow, or how their molecular composition is regulated. Cell-based experiments have led to conflicting observations, and we have limited tools to understand how changing molecular composition can create focal adhesions with specific chemical or physical characteristics that alter downstream signaling. To address these important questions, Dr. Case has developed a novel biochemical reconstitution of focal adhesions using purified proteins on supported lipid bilayers. This work identified seven proteins that are sufficient to form focal adhesions through liquid-liquid phase separation. Studying integrin-dependent mechanotransduction through the lens of phase separation could drive significant advances in the field. The Case Lab will use a variety of experimental strategies to understand different aspects of integrin-dependent mechanotransduction. They will directly test different models of mechanotransduction with biochemical reconstitution and confirm the importance of any new in vitro observations with cell-based assays. They will investigate how focal adhesions mature, how forces are transmitted across focal adhesions, and how the biochemical composition of focal adhesions is regulated. This project will take advantage of a novel experimental approach to challenge the current dogma about integrin-dependent mechanotransduction, and will reveal how specific molecules regulate focal adhesion growth and composition.

NIH Research Projects · FY 2024 · 2022-09

When learning a new task, both rats and humans exhibit suboptimal behaviors plagued with superstitious ticks and idiosyncratic biases. One prominent example of such suboptimality are sequential effects: animals tend to bias their choices based on previous decisions and outcomes, hindering performance in common laboratory tasks using independent trials. Recurrent neural networks (RNN) have become a common tool to study potential neural mechanisms of cognition. Yet, RNNs typically behave much closer to optimality in laboratory tasks than real subjects. We suggest this behavioral difference is rooted in the fundamental discrepancy between how animals and current RNNs learn: unlike animals before learning, RNNs before training are tabula rasa and their connectivity is adjusted exclusively to the local contingencies of the task. We hypothesize that animals’ learning of simple laboratory tasks builds mostly on pre-existing programs, namely structural prior, that have been shaped by evolution for the species’ fitness in a given ecological niche. Sequential effects are a manifestation of such pre-wired strategies, which may ultimately support learning. To test this, we will characterize sequential effects during learning of a set of perceptual tasks and identify their underlying neural circuitry. We will compare animals’ behavior with RNNs which, after being equipped with structural priors, can mimic the animal’s ability to learn new tasks. Objectives Objective 1. Compare sequential effects in humans and rats with those developed by RNNs. Objective 2. Characterize the role of the corticostriatal circuit mPFC --> DMS in the tasks and the site of plasticity necessary for task learning. Objective 3. Characterize the neural mechanisms underlying the representation of relevant variables in the brain of the rat and in RNNs.

NIH Research Projects · FY 2024 · 2022-09

Project Summary and Abstract Gene therapy enables the treatment of a large number of genetic diseases through delivery of nucleic acids striking at the root of the disease. This is advantageous because it is highly modular, allowing for a number of different cargo nucleic acids to be delivered depending on the disease cause. As such, the ideal gene therapy delivery vector would be able to carry a variety of cargo, deliver this in a targeted manner, and accommodate a range of cargo sizes. There are a number of techniques utilized to deliver nucleic acids including viral systems like adenovirus, adeno-associated virus (AAV), and lentivirus, as well as non-viral methods including nanoparticles. Although these therapies can be successful, a key limitation to currently used vectors is the immune response which can lead to ineffective delivery of nucleic acid cargo. There is currently a need to develop effective and non-immunogenic delivery vehicles for gene therapy for a wide range of diseases, including neurological disease, for which effective delivery vehicles have yet to be designed. To this end, mammalian genomes contain numerous virus-like genes, some of which have been co-opted by their host cells for important functions. Among these are homologs of gag, which encodes the capsid protein. We hypothesize that endogenous genes encoding a capsid domain have the ability to self-assemble into capsids and mediate intercellular communication by binding, secreting, and delivering nucleic acid cargos. We propose to explore and re-engineer endogenous capsid-containing proteins for use as gene therapy vectors. We hypothesize that delivery vehicles composed entirely of self proteins will be more effective than standard vectors as they could be non-immunogenic. Here we propose to use an approach combining in vitro characterization, re-engineering, and in vitro and in vivo validation to identify candidate proteins and learn how they can be re-engineered. These systems will ideally be modular, having both programmable cargo and tropism to treat a range of diseases. We hope that by identifying and re-engineering these systems, the resulting fully endogenous delivery vehicle will be useful for efficient, reprogrammable, and non-immunogenic gene delivery. With the goal of becoming an independent investigator, this project will also support development of computational biology skills, molecular biology expertise as well as mentorship and scientific communication skills. These will be supported by the excellent research environment at the Broad Institute and MIT.

NIH Research Projects · FY 2024 · 2022-09

Implantable interfaces for neuromodulation is necessary to advance fundamental neuroscience research, develop new treatments for neurological disorders, and create efficient breakthrough neuroprosthetics. However, modern implants based on multi-electrode arrays suffer from low spatial resolution, high invasiveness with complicated implantable procedures, the need for a chronic opening for connecting wires, and substantial foreign body reaction, eventually leading to device failure. On the other hand, various groups have developed nanoparticles-based transducers that can wirelessly modulate neurons with high precision when actuated with external stimuli. Nevertheless, nanoparticles hold several disadvantages due to their small size (resulting in neurotoxicity, migration, aggregation, etc.), restricted fabrication procedures, and limited design or integration opportunities. Hence, minimally invasive and non-genetic technology that can enable wireless neuromodulation with high spatio-temporal resolution and stable interface remains an unmet goal till date. Therefore, we propose to develop an innovative thin-film-based structure able to wirelessly influence the neuronal membrane to induce or inhibit action potential propagation along a specific path of connected neurons. These devices will be designed and produced with subcellular dimensions to be injected into the neural tissue, diffuse, and wrap around axons and dendrites (creating conformable and stable neural interface); hence, they are named nanoCUFFs. The nanoCUFFs will be composed of two types of polymers: i) an azobenzene polymer for photo-induced reconfiguration of thin films rolled into microtubes, accommodating single axons; and ii) a semiconducting polymer for transduction of light pulses into stimuli for neuronal opto-modulation. Polymers allow creating soft, biocompatible, and conformable structures for a minimal mismatch and maximal coupling with the biological tissue. Once the nanoCUFFs are produced and characterized, we will verify their wrapping capabilities around axons and dendrites, neuromodulation efficiencies as well as ability to influence distinct selected subpopulations of neurons (using micro-patterned light) in neural cultures. The ability to engineer the nanoCUFFs’ material composition and photo-induced effects on a thin-film platform favors the future integration of nanoelectronics components for additional functionalities. For instance, multiplexing and sensing devices could be developed for smart closed-loop neuromodulation. This technology can simultaneously achieve ultra-low invasiveness, high-spatio-temporal precision, selectivity and stable junction with cells and thus, is highly promising for not only fundamental neuroscience but also novel therapeutics.

NIH Research Projects · FY 2025 · 2022-09

Modified Project Summary Section Bioelectronic implants provide a versatile platform for diagnosis, therapeutics as well as basic research but require invasive surgery. Here, we propose a paradigm shift: the ‘Circulatronics’ technology, wherein ultra-small bioelectronic devices target desired regions in the body for sensing and treatment, without the need for surgery. Its realization requires: i> nanoelectronic devices that are aggressively miniaturized (to fit inside vasculature) and extremely low power (to work deep inside body with low harvested energy); ii> heterogeneous integration of power-source and nanoelectronic circuits in a single device platform; iii> targeting the diseased regions for implantation without surgery. Accomplishing these requires innovations in diverse fields of applied physics, nanoelectronics and bioengineering and we are uniquely enabled due to our expertise in not only physics and solid-state nanoelectronic devices but also in bioelectronics, synthetic biology and neural engineering. We will build upon our work in developing ultra-scalable and record-low power nanoelectronics, which can lead to beyond-Silicon dimensional scalability to achieve i) and create sub-cellular sized and highly energy-efficient nanoelectronic devices. Moreover, we will leverage our research in building novel van der Waals heterostructures employing heterogeneous material systems enabled by atomically thin 2D materials to accomplish ii). For achieving iii), we will explore different surface functionalization techniques and leverage our expertise in synthetic biology. Circulatronics is a radical technology which can change the landscape of the field of biomedical implants and transform bioelectronic medicine. By alleviating surgery, it not only offers ultra-low invasiveness but can extend healthcare to patients not suited for surgery. These devices can modulate biological signals and can also integrate sensing functionalities. Since they can reach every nook and cranny of the body, they can obtain information from and treat intricate regions in body, which cannot be accessed by other technologies. Moreover, being extremely small, they can interact at a single cell or even subcellular level, to provide highly precise diagnosis and therapeutics as well as fundamental insights into biology.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Chronic itch affects 13% of the population and is associated with over $90 billion in annual population- expenditures in the US. It has a profound negative impact on quality of life, and is often as debilitating as chronic pain. Yet, there are currently no FDA-approved treatments for chronic itch. A major obstacle in assessing therapeutics for itch is the difficulty in measuring it, which hinders assessment of outcomes in the clinic and the development of new drugs. The current clinical standard for quantifying itch relies on patients’ self-assessment of the severity of their itch on a scale of 0 to 10, which is: 1) highly subjective and hard to generalize across patients, 2) lacks sensitivity to small changes, and 3) is difficult to use in vulnerable populations such as children and those with cognitive impairment. Thus, clinical research on itch has an urgent need for a new objective, accurate, and low overhead method for quantifying itch. Furthermore, given that disturbed sleep is a major factor leading to diminished quality of life for chronic itch patients, the new method should ideally also assess sleep quality. The overall objective of our proposal is to provide an objective, sensitive, and reliable metric for measuring both itch and its impact on sleep. The central hypothesis of this proposal is that a novel, wireless sensor can be employed to effectively capture scratching activity and associated itch morbidity, and also measure its impact on sleep. Our approach is based on a non-obtrusive wireless device that sits in the background at home, much like a Wi-Fi router. It analyses the radio signals that bounce off people's bodies using novel machine learning models to infer people’s sleep quality and scratching motion -- and it does it in a touchless manner without asking patients to wear sensors, or incur any burden. The Katabi lab invented this sensor technology and has already demonstrated its ability to measure sleep stages, respiration signal, heart rate, falls, gait and other behaviors in humans. Further, the Katabi and Kim labs have preliminary data that demonstrate the feasibility of extending this method to monitor scratching in a touchless manner in chronic itch patients. The specific aims of this proposal will assess the accuracy, sensitivity, and specificity of this novel method in measuring nocturnal scratching in chronic itch patients, its performance in comparison to the current clinical standard based on patients’ self-assessment of their condition, and its ability to track changes over time in the same patient. It will also leverage the device’s ability to monitor sleep to assess the impact of itch on patients’ sleep quality, and the relationship between sleep metrics (e.g., sleep onset, sleep efficiency, and sleep stages) and scratching severity. The rationale for this proposal is that the ability to quantify itch and its impact on sleep in an objective, sensitive method that is widely applicable, including to children and cognitively impaired patients, would improve clinical research, and facilitate the assessment of therapeutics for both disease management and drug development.

NIH Research Projects · FY 2025 · 2022-09

Environmental health and genomics are two academic subjects that receive little attention by institutions that train health professionals. Unfortunately, many health professionals remain unaware that an individual’s genetic makeup can influence their susceptibility to an environmental hazard. This MIT led program will address health professionals’ need for training in genomics and environmental health with a one-week Short Course on gene-environment interactions. MIT instructors working in collaboration with NYU and UNM, as well as with MBIRI, a NIH research hub in S. Dakota, will be hosted by local universities serving both rural and urban regions of the country for this Short Course. The program will recruit course participants who are science educators and as well as health professionals, as science educators offer a multiplier effect when sharing the course information in their classes. Overall, the Short Course faculty plans to partner with rural colleges in the mid-west, such as South Dakota Black Hills State University, as well with urban/suburban schools such as the Ramapo College of New Jersey with their nursing programs. The participants will start each day with a lecture/discussion on a pre-selected, community-relevant environmental health topic. Lectures will be followed by an engaging hands-on program using MIT-patented DNA and Protein models. Pre- and post- tests will be used to measure and record course participants’ learning gains from the hands-on activities. In summary, the Short Course participants will have learned about gene-environment interactions through specific examples. They will also have created an environmental health messaging product on a health topic of their own choosing and shared this in a presentation in class during the course. Additionally, three months after each Short Course a follow up study will be conducted via email to collect feedback on how our participants have utilized or shared their new knowledge about gene-environment interactions. The R25 Short Courses will fill a much-needed gap in health care professionals’ training, bringing an awareness of gene-environment interactions to improve public health delivery and enhance the public’s quality of life.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS) are irreversible and currently incurable neurodegenerative diseases with more than 65,000 new cases in the USA each year. Their core motor symptoms are respectively caused by dysfunction and death of dopaminergic neurons within the substantia nigra and motor neurons in the cortex, brainstem, and spinal cord. However, non-cell-autonomous contributions to disease progression are widely recognized and include cerebrovascular (CV) dysfunction. The CV is formed by several highly specialized cell populations, including brain endothelial cells (BECs), mural cells, fibroblasts, and glia. Given the CV’s critical role in regulating biomolecule transport into and out of the brain, blood flow, and responses to physical or chemical stress, understanding the molecular underpinnings of early CV changes during PD and ALS may be critical to develop disease-modifying treatments. Prior work indicates that CV changes can occur during the progression of PD and ALS, including leakage of the blood-brain barrier (BBB), angiogenesis, dysfunctional efflux activity, dysregulated blood flow, and increased immune cell trafficking. However, findings from brain imaging (MRI) and histological analysis are not inclusive of all CV functions nor able to identify transcriptional regulators, while studies using animal models are not representative of sporadic human disease which accounts for ~90% of PD and ALS cases. In this proposal, I will characterize cerebrovascular dysfunction during sporadic PD and ALS with cell type-specificity and whole genome-resolution from post-mortem tissue, and will benchmark the degree to which this dysfunction is recapitulated by iPSC-derived in vitro models. This work is grounded in recent application of blood-vessel enrichment (BVE) and single nucleus RNA sequencing (snRNA-seq) approaches to profile gene expression of CV cells, and the development of transcription factor overexpression-based differentiation of BECs from induced pluripotent stem cells (iPSCs). In Aim 1A, I will conduct snRNA-seq on blood vessel enriched substantia nigra from post-mortem PD patients and age-matched healthy controls, and will then validate cell type-specific dysfunction using immunofluorescence and in situ hybridization studies. In Aim 1B, I will differentiate BECs from PD patient iPSCs and age-matched healthy controls and then conduct snRNA-seq to determine how post-mortem hallmarks of dysfunction are reflected in vitro. In Aim 2, I will take a similar approach by conducting snRNA-seq on ALS patients blood vessel enriched motor cortex and iPSC-derived BECs compared to healthy age-matched post-mortem tissue and iPSC controls. By characterizing CV gene expression using cutting-edge single nucleus profiling of PD and ALS post- mortem tissue and iPSC-derived models, this proposal will identity previously unrecognized mechanisms of CV dysfunction and serve as a critical launchpad for future studies to test causality in disease processes and validate therapeutic targets across in vivo and in vitro models.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Extracellular polysaccharides play critical roles across all domains of life. Bacterial polysaccharides are a diverse class of macromolecules with multiple biological functions, including mediating interactions with the external environment and preserving cell wall integrity. Bacterial glycosyltransferases are responsible for polysaccharide diversity through their differences in substrate specificity and linkage production. Polysaccharide biosynthesis and elongation can occur by multiple mechanisms; the least understood is processive polymerization. Processivity is elicited from an enzyme’s ability to retain the acceptor through numerous elongation steps. This process reduces the production of short-length polysaccharides, which could be harmful to bacterial fitness. Processivity may represent a common and critical mechanism for polysaccharide biosynthesis and length control. Production of the mycobacterial galactan by galactofuranosyltransferase 2 (GlfT2) was shown to proceed by a processive mechanism. The galactan of Mycobacterium spp. is an essential structural glycan, functioning as a component of the cell wall structure of human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Galactan truncation decreases cell fitness, promotes periplasm thinning, and increases antibiotic susceptibility. Therefore, enzymatic processivity by GlfT2 likely ensures the galactan is of sufficient length. The proposed studies seek to define the mechanism of GlfT2 processivity and the biophysical parameters that dictate product length distributions. This project encompasses training in enzyme production and characterization, enzyme kinetics assays, and enzyme structure determination. The Kiessling group, leaders in chemical glycobiology, and the Department of Chemistry at MIT provide a rich environment to acquire these research skills. The research environment also offers opportunities to engage in science communication, literature analysis, and career development. The results from the investigations proposed are expected to provide a framework for mechanistic analysis of processive glycosyltransferases found in other bacteria and across the different domains of life. New insights into this under-characterized class of enzymes will provide novel targets to combat the modern emergence of antibiotic-resistant bacteria.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT The discovery of potent pharmaceutical agents requires expedient access to a wide range of diverse molecular architectures, and the chemical tools available to the medicinal chemist both enable and limit this venture. Over the past half century, transition metal-catalyzed cross-coupling has grown into a powerful strategy for organic synthesis. However, the use of the d-block elements presents specific disadvantages, including low acceptable metal content in pharmaceutical products and susceptibility to unproductive coordination by polar medicinally- relevant functional groups. Thus, there has been a recent surge in interest in developing cross-coupling catalysts containing the naturally abundant main group elements of the p-block. Mechanistic understanding of the reactivity of main group catalysts lags far behind that of transition metal catalysts, and synthetic applications remain limited. One particularly promising approach for main group catalysis is to utilize the P(III)⇌P(V) redox couple as one would employ the analogous redox couples of transition metal catalysts. To develop improved biphilic catalysts for phosphorus redox cycling chemistry, a more complete mechanistic understanding of the factors affecting catalyst performance is necessary. Towards this end, the proposed research will employ two distinct approaches to catalyst development: multivariate regression analysis to correlate phosphetane structure with desired redox properties, and computational modeling to guide the rational design of a novel class of boron- and silicon-containing phosphetanes with reduced frontier orbital energy gaps. The detailed study of these catalysts will provide valuable insights into the ability of phosphorus-based catalysts to facilitate carbon- heteroatom bond formation, enabling the development of an allylic amination reaction of immediate medicinal relevance. This research proposal supports and aligns with the fellowship goals by requiring new skills to be learned in inorganic synthesis, mechanistic study, and computational modeling that complement previous training in synthetic organic methods development. The Radosevich lab provides an ideal research environment uniquely suited to facilitate training in these areas, as evidenced by their pioneering efforts in the development of phosphorus-catalyzed reactions. Prof. Radosevich’s personal commitment to supporting postdoctoral researchers in their development into independent investigators ensures that the professional training goals will be achieved. Lastly, MIT, as one of the most productive research institutions in the world, provides the resources and equipment necessary to carry out the research proposed.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT Spinobulbar Muscular Atrophy (SBMA) is an incurable neurodegenerative disorder that is characterized by the toxic accumulation of mutated androgen receptor (polyQ-AR) proteins. The molecular chaperone protein DnaJB6 is specialized to prevent polyQ-AR aggregation in cells and suppress disease phenotypes by preventing polyQ-AR aggregation and recruiting another chaperone protein, Hsp70 to polyQ-AR. While DnaJB6 represents an exciting target for developing SBMA chemotherapeutics, its dynamic protein-protein interactions and complex structure present significant challenges for efforts to discover and design DnaJB6 chemical ligands. DnaJB6 associates with itself to form complexes that appear to exchange between larger and smaller oligomeric states over time. I hypothesize that binding to either polyQ-AR or Hsp70 causes changes in the stability of DnaJB6 complexes, and that these changes in DnaJB6 dynamics can be exploited to discover chemical probes. DnaJB6 chemical probes could then be used to probe pathological aggregation in SBMA. To test this hypothesis, I will characterize DnaJB6 complex stability and discover chemical matter that tunes DnaJB6 activity in the K99 phase of this award. In the R00 phase, I will use these molecules to probe SBMA in cell and animal models. This work is significant because the tools resulting from my studies will not only have applicability for studying SBMA, but also for other neurodegenerative disease where DnaJB6 can suppress protein aggregation (i.e. Huntington's Disease, Spinocerebellar Ataxias, and Parkinson's Disease) and chaperone biology more broadly. My proposed studies are innovative, as they will yield the first DnaJB6-targeted chemical probes and a novel strategy to understand the molecular underpinnings of SBMA.

NIH Research Projects · FY 2024 · 2022-08

PROJECT SUMMARY/ABSTRACT Intracellular bacterial pathogens manipulate host cells through a vast array of mechanisms. Studying these interactions has propelled our understanding of therapeutic development against these pathogens and host cell biology. However, many intracellular pathogens cannot be easily studied due to their obligate nature and resistance to genetic manipulation. These include the spotted fever group (SFG) Rickettsia, which cause a range of potentially severe arthropod-borne human illnesses, including Rocky Mountain spotted fever. The development of new random mutagenesis systems for SFG Rickettsia has propelled studies of these microbes and hinted at the remarkable diversity of unprecedented pathogen innovations in this genus. Recently, our lab performed a small-scale transposon mutagenesis screen in the model rickettsial species R. parkeri to identify attenuated mutants. This screen led to the isolation of >100 R. parkeri mutants with infection defects, with only a few containing insertions in genes previously linked to R. parkeri virulence. The remaining strains represent a valuable tool for probing and understanding R. parkeri and intracellular pathogen biology. Over 15% of the genes hit in this screen are unannotated. Two of these unannotated genes, hrtA and sp50, encode R. parkeri proteins that are predicted to be surface-exposed or secreted and have putative structural features suggestive of direct binding to host proteins. I hypothesize that HrtA and Sp50 are novel R. parkeri secreted or surface-exposed effectors that can hijack specific host functions to promote infection. In this proposal, I will first demonstrate the spatiotemporal niches of both HrtA and Sp50 (Aim 1) to establish how they phenotypically contribute to R. parkeri infection. Then, I will use affinity purification approaches to identify direct host-derived interactors of HrtA and Sp50 (Aim 2). Finally, I will use host-direct genetic perturbation screens to profile host-pathogen synthetic genetic interactions with R. parkeri strains lacking HrtA or Sp50 (Aim 3). Through this work, I will not only extend our understanding of SFG Rickettsia pathogenesis, but will also demonstrate the potential of a synthetic genetic approach for investigating and annotating pathogen genes of unknown function. Results from these studies may also inform development of therapeutics such as vaccines against SFG Rickettsia species. The training environment at MIT, where this project will be carried out, is outstanding and highly collaborative. All facilities and equipment required for this project are available to the applicant (Dr. Brandon Sit). The training plan accompanying this project involves the joint mentorship of Dr. Sit by Drs. Rebecca Lamason (primary sponsor) and Paul Blainey (co-sponsor), and is designed to position Dr. Sit for a transition to an independent investigator position at the end of this work.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Alzheimer’s disease is a devastating dementia with no known cure. While research has advanced our knowledge of the genetics and molecular biology of AD, it is not yet known why some areas of the brain are affected, while others are spared. Additionally, the sensitivity of circuits and synaptic connections in disease progression are not known. We will examine the connectivity of the locus coeruleus (LC), which projects to most areas of the brain, and is one of the regions to show pathology earliest in AD. We will characterize the populations of these neurons based on their connectivity and sensitivity to degeneration with aging and in AD model mice, both from a global connectome level and with single-cell approaches for molecular signatures. Additionally, we with look in more detail at the entorhinal cortex, one of the recipients of LC connections with preferential cell loss, and characterize the specific populations and input/output relationships in response to aging and LC pathology.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY The molecular and cellular heterogeneity of senescent cells remains poorly characterized. The knowledge gap is mainly due to the lack of proper technology to characterize the cell states, types, and circuits in intact tissues. Thus, we will need novel technologies to map the multidimensional parameters of senescence across diverse tissue environments at molecular, cellular, and morphological levels and over longitudinal time frames. Single cell multi-omics and molecular profiling assays (e.g., single-cell RNA-seq, single-cell ATAC-seq, single-cell proteomics, methylomics, metabolomics) have opened new windows into understanding the properties, regulation, dynamics, and function of cells at unprecedented resolution and scale. However, these assays are inherently destructive. Cells need to be dissociated, fixed, or lysed for these molecular profiling assays. Raman microscopy offers a unique opportunity to comprehensively report on the vibrational energy levels of molecules in a label-free, nondestructive manner with subcellular spatial resolution. With recent advances in Raman microscopy, single-cell and spatial multi-omics, and machine learning, we have developed “Raman2RNA” (R2R), an experimental and computational framework to infer single-cell expression profiles in live cells through label- free hyperspectral Raman microscopy images combined with multi-modal data integration and domain translation. In this proposal, we aim to develop “SenNetRaman”, an innovative experimental and computational platform to character the molecular heterogeneity of senescent cells through label-free hyperspectral Raman microscopy, single cell and spatial genomics, and machine learning. In the UG3 phase, we aim to develop “SenNetRaman” for characterizing single cells in lung tissues corresponding to young, naturally aged or stress- induced senescence states from well-established mouse models. We will develop a high-throughput Raman microscopy system for label-free characterization of the molecular heterogeneity of senescent cells and identify Raman signals/markers predictive of gene expression and corresponding to various senescent cell states and types. In the UH3 phase, we will demonstrate “SenNetRaman” for characterizing senescent cells across multiple senescence model systems including human lungs, brains, and skins from an established human senescence tissue mapping center. Overall, “SenNetRaman” is a modular and universal framework to link imaging data with single-cell multi-omics data for building quantitative biomolecular tissue maps of human senescent cells. Our application is innovative in the approach to study senescence by leveraging the recent advances in imaging, single-cell genomics, and machine learning. The results of this project will help identify novel markers and reveal new biology of senescence. “SenNetRaman” builds upon the SenNet Initiative and can be readily adapted to existing NIH single-cell tissue mapping efforts, including the Human Tumor Atlas (HTAN), Human Biomolecular Atlas Program (HuBMAP), and Human Cell Atlas (HCA) that will transform future biomedical and clinical research.

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract Immediately after fertilization, metabolism in the early embryo undergoes drastic remodeling. While studies have thoroughly characterized pre-implantation embryonic development, measurements of metabolism in the embryo post-implantation, from gastrulation to birth have been limited by scarcity of tissue and inaccessibility of the embryo. in vitro differentiation of human embryonic stem cells provides an unlimited source of tissue and a platform to study the metabolic transitions that occur within the rapidly expanding embryo. Using directed differentiation of human embryonic stem cells into pancreatic epithelium, this proposal aims to combine metabolic interrogation of cell proliferation and biomass accumulation with developmental biology. These studies will blend recent conceptual advances in cancer metabolism with an in vitro differentiation platform to understand how metabolism instructs and directs cell fate decisions in the developing fetus. Experiments will first focus on cellular redox state, testing whether changes in NAD+/NADH ratio as human embryonic stem cells undergo differentiation into pancreatic epithelium are necessary to support differentiation into the pancreatic lineage. Then, kinetic tracing studies using isotopically labeled nutrient sources will quantify rates of metabolic flux to determine how changes in overall metabolism contribute to differentiation. Finally, metabolites that have been identified to undergo large fluctuations in abundance throughout differentiation will be studied to determine whether their accumulation is necessary for successful pancreatic differentiation. This proposal is designed to explain the roles of metabolism in the developing pancreas as it continuously differentiates and expands in a changing nutrient environment. Results from each of these aims can be used to improve in vitro differentiation of stem cells by providing new mechanisms to guide successful differentiation into a desired cell type. By providing new insight into metabolic regulation of stem cell fate, this work will enhance the ability to generate curative stem cell-based therapies for patients suffering from lost or dysfunctional tissues, especially type 1 diabetes. These therapies hold great promise for improving health and quality of life for millions of patients. These experiments will be carried out in the laboratory of Professor Matthew Vander Heiden within the Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology. Training under this fellowship will include presentation of this data at institutional and public conferences, publication of this work in peer-reviewed scientific journals, and regular meetings with professor Vander Heiden to prepare the applicant for success as a tenure track research faculty member.

NIH Research Projects · FY 2025 · 2022-07

Abstract/Summary The U.S. is currently facing a drug addiction (DA) epidemic while there is a dearth of innovative technologies for preventing, diagnosing and treating DA and substance use disorders (SUDs). The program on Entrepreneurship and Innovation for Biomedical Product Development (EI4BPD) aims to advance the inception and development of products and services to develop, scale, and deliver effective prevention, diagnostic and treatment solutions for substance use disorders by training life science researchers in entrepreneurship, innovation, biomedical product development and commercialization. Specifically, this program will: 1) Combine the expertise of entrepreneurship, innovation, DA, and education scholars toward a customized and unified curriculum in entrepreneurship, early- stage biomedical technology commercialization and biomedical product development. 2) Recruit 4 consecutive yearly cohorts of 15 fellows, who are U.S. scientists working in the fields of SUDs research, to receive, free of charge the training on how to foster the development of early-stage biomedical technologies and how to advance their technologies from the research and academic laboratory into the commercial world. The selection will occur through a highly competitive process. 3) Implement the developed curriculum as an educational program consisting of tailored online (live and self-paced) and in-person educational activities driven by coaches from academia, government and industry. 4) Create a Community of Peers for all SUDs researchers and industry experts to provide program sustainability and a space for ideas’ cross-pollination. The educational program will consist of a kickoff event to promote community building and set the program’s goals. It will be followed by two online courses on innovation and entrepreneurship covering user understanding and technology evaluation. The courses will be complemented by tailored live webinars and online support activities. Finally, the program will be concluded with an in-person hands-on bootcamp on entrepreneurship and innovation for biomedical product development facilitated by practicing biomedical entrepreneur and pharmacotherapy coaches. Each new cohort will be supported by a variety of industry experts, speakers and coaches that will be part of a Community of Peers, which will grow every year to keep all alumni connected to support future biomedical product development efforts.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Multicellular organisms use intercellular communication to coordinate cell function and maintain tissue homeostasis. Recent work suggests that this communication is driven in part by the exchange of organelles (via trans-endocytosis) and mechanical cues directly across cell-cell junctions. Dysregulation of this communication leads to cancer and cardiovascular diseases. Despite its importance, we lack a fundamental molecular understanding of how intercellular communication occurs because of the limited number of cell biological tools capable of probing the molecular mechanisms at cell-cell contacts. This proposal seeks to elucidate the regulatory mechanisms of the pathways thought to control intercellular communication by studying how they are manipulated when under microbial control. The bacterium Listeria monocytogenes disseminates through human tissues using a process called cell-to-cell spread, which is a vesicular-mediated form of intercellular exchange that mimics host trans-endocytosis. Listeria spreads from cell to cell by mobilizing the host’s actin cytoskeleton for intracellular motility and transport to the cell-cell junction. Once at the junction, it pushes against the membrane and forms a double-membrane protrusion that is engulfed by a neighboring cell. Studying this distinctive spreading process will allow us to examine several outstanding cell biological questions. First, are specific endocytic pathways used at cell-cell junctions to engulf large cargo like microbes? Second, are mechanically-sensitive membrane domains or membrane curvature proteins activated as Listeria pushes against the junction during spread? To answer these questions, we used a high-content, image-based siRNA screen to test if Listeria requires host intercellular communication pathways during spread. We discovered that the endocytic and mechanoresponsive caveolar proteins CAV1, CAV2, and PACSIN2 promote Listeria spread. We also revealed a putative role for 19 other host proteins, including those that regulate membrane curvature, trans- endocytosis, and adhesion. Our preliminary findings suggest the overall hypothesis that Listeria subverts multiple intercellular communication pathways to promote cell-to-cell spread. In Aim 1, we will determine how PACSIN2 and caveolins coordinate their activities to promote the engulfment stage of cell-to-cell spread. In Aim 2, we will reveal which of the remaining hits regulate Listeria spread specifically, how they function, and if they work independently or together with caveolae. In the end, our proposed studies will improve our fundamental understanding of host-microbe interactions and basic cell biology, and may uncover how intercellular communication goes awry in human disease.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY Infectious diseases such as HIV, malaria, tuberculosis, and seasonal influenza epidemics and emergence of new pandemics remain major global health problems highlighting the need for innovative approaches in vaccine design. The precise kinetics of antigen exposure relative to inflammatory cues is known to play a critical role in shaping a coordinated cellular and humoral immunity and thereby enhancing the vaccine immunogenicity and efficacy. Current vaccination strategies, however, do not include mechanisms for the temporal control of antigen and adjuvant exposure to lymphoid tissues. Here, we propose incorporating synthetic biology approaches to create nucleic acid-based vaccines where the kinetics of vaccine (antigen and adjuvant) exposure can be controlled using orally-available FDA-approved small molecule drugs. This strategy is enabled using self-replicating RNAs termed replicons that encode antigens and cytokine molecular adjuvants and encompass regulatory mechanisms governed by the FDA-approved small molecule drug, trimethoprim (TMP). This strategy allows the delivery of replicons encoding antigens and cytokines in vivo with a single bolus injection and then controlling the amplitude and duration of antigen and cytokine expression by oral administration of TMP. Using the RNA replicon platform provides several advantages: (i) it allows antigens and cytokines to be produced in their native conformation; (ii) it self-replicates and therefore persists inside the cells longer than mRNA and sustains a steady supply of “fresh” antigen and adjuvant; (iii) unlike DNA therapeutics, it does not harbor the risk of genome integration and also does not require delivery to the nucleus for transgene expression. An HIV envelope immunogen, known as the engineered outer domain (eOD-GT8), will be used as the model antigen, and interleukine-2 and interleukine-12 will be used as the cytokine molecular adjuvants. The Specific Aims of this project are: (1) Generate small molecule-responsive RNA replicon vaccines enabling control over the dynamics of antigen and adjuvant expression. (2) Identify optimal temporal patterns of antigen and adjuvant exposure maximizing the protective immunity elicited by replicon vaccines. Results from this project will establish a vaccine platform that allows modulating and promoting the magnitude and quality of T cell and antibody responses following immunization by taking a drug available as an oral pill, as a simple and clinically- translatable strategy to enhance vaccine-induced immunity. In addition, elucidating the optimal cytokine and antigen exposure patterns that confer protection will provide critical information and insights for use in conceiving better vaccine design strategies.

NIH Research Projects · FY 2025 · 2022-05

NMR-Based Rapid Fluid Assessment: Device Design and Signal Processing PROJECT SUMMARY Our goal is to develop a portable, non-invasive, measurement of volume status to improve quality of life and reduce morbidity and mortality among hospitalized patients. Maintenance of euvolemic status and proper fluid balance are critical for health and improved outcomes for renal, cardiovascular, and many other disease types as well as in healthy populations prone to dehydration such as athletes and soldiers. Our laboratory has previously constructed a portable single sided magnetic resonance (MR) sensors that is capable of resolving individual fluid compartments (subcutaneous, intramuscular, etc.) within tissue. This sensor was used in a pilot clinical trial with end stage kidney disease patients. Quantitative MRI results on these patients demonstrated that the first sign of fluid overload among hemodialysis patients is an expanded skeletal muscle extracellular fluid (ECF) space. The early stage nature of the technology was evident in that study as the portable sensor could not unambiguously differentiate skeletal muscle tissue from subcutaneous tissue. The proposed research includes the design of a new portable low-field MR sensor and improved signal processing that will allow it to capture the same quantitative assessment of volume status currently achievable with quantitative MRI (qMRI). We measure local fluid distribution in the target in vivo tissue compartment. In the case of fluid volume status, our hypothesis is that a localized skeletal muscle measurement is representative of systemic fluid distribution based on results from a prior study1. The optimized sensor will have the sensitive region of the magnetic field designed to target the skeletal muscle. The existing and newly designed portable MR sensors will be used to measure intramuscular fluid distribution in end stage kidney disease patients undergoing dialysis treatment as well as in healthy athletes experiencing exercise induced fluid loss. The sensor measurement will allow for quantification of volume overload (hypervolemia) or depletion (hypovolemia) and allow for its use in clinical decision making. Dialysis patients and athletes are the target population for the following proposal. The rapid reduction in fluid volume during a dialysis session and exercise respectively provides the ideal clinical context for performing repeated fluid status measurements.