Massachusetts Institute Of Technology

NIH Research Projects · FY 2025 · 2021-02

The current proposal describes experimental approaches to determine how neurons regulate structural and functional maturation of active zones (AZs), a key signaling hub where synaptic communication occurs. Although membrane trafficking mechanisms are highly conserved across cells, additional synapse-specific regulation has evolved to mediate rapid Ca2+- dependent synaptic vesicle (SV) fusion at specialized presynaptic AZs that are precisely aligned to postsynaptic receptors. Multiple evolutionarily conserved proteins are found at AZs, including RIM, RIM binding protein, Syd-1, Liprin-α, ELKS/CAST/Bruchpilot (BRP), Bassoon/Piccolo/Fife and Unc13. Previous studies in our lab demonstrated that the hundreds of AZs formed by a single glutamatergic motoneuron in Drosophila have a heterogeneous distribution of synaptic strength, with neighboring AZs often showing >50-fold differences in the probability of release (Pr) of SVs. We found that AZ maturation drives increased synaptic strength occur over a multi- day developmental period, with newly formed AZs developing as weak Pr sites before maturing into high Pr AZs through the coordinated accumulation of a core set of proteins. In the current application, we will determine how neurons regulate structural and functional maturation of AZs, and how variations in these processes drive synaptic diversity. The mechanisms regulating AZ maturation fall into two broad categories: those that control cell-wide availability of key building blocks to growing AZs (Aim 1) and those that affect capture and retention of new material at individual AZs (Aim 2). We will determine whether specific AZ proteins are produced and transported in excess of their incorporation into growing AZs, or whether their availability at the synaptic terminal is rate-limiting for AZ maturation. In addition, we will characterize the efficiency of material capture at individual AZs throughout the AZ maturation cycle. Finally, we will examine how material availability and capture differ in tonic and phasic motoneurons that innervate the same postsynaptic muscle, but display striking differences in their AZ organization and SV release properties (Aim 3). These studies will provide new insights into how synaptic strength develops across the cohort of AZs of a neuron, as well as how synaptic diversity can be more broadly controlled across neuronal subclasses.

NIH Research Projects · FY 2025 · 2021-02

Prostate cancer is the most common cancer in the US male population, with an estimated 160,000 new cases in 2018. Treatment with radical prostatectomy (RP), complete surgical excision of the prostate, results in favorable oncologic outcomes with long-term survival benefits. Nerve-sparing RP is favored if cancer does not involve the neurovascular bundles since patients have better recovery of sexual function and continence, major factors determining postoperative quality of life. However current preoperative methods do not accurately identify patients who could be treated by nerve-sparing RP. The NeuroSAFE study, Schlomm, et al., 2012 demonstrated that comprehensive intraoperative frozen section analysis (FSA) of margins near the neurovascular bundles increased the rate of nerve-sparing RPs. However comprehensive intraoperative FSA required extensive time and personnel, which is impractical for most hospitals. Nonlinear microscopy (NLM) can generate images of freshly excised tissue resembling H&E histology, without freezing or microtoming, reducing the time and labor required for pathology evaluation. We developed custom NLM technology and specimen handing/staining protocols for rapid, high-throughput evaluation of prostatectomy specimens. Our preliminary data demonstrates that NLM detects prostate cancer with 97% sensitivity and 100% specificity compared to formalin fixed paraffin embedded (FFPE) H&E in a study of 122 RP specimens from 40 patients with blinded reading by three pathologists. NLM promises to enable intraoperative evaluation of RP specimens with a simplified workflow that is practical for widespread clinical adoption. Our hypothesis is: NLM can be used to rapidly assess prostate surgical specimens and increase nerve-sparing RP rates without increasing positive margin rates. This is a collaborative, multidisciplinary program with investigators at the Massachusetts Institute of Technology and Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School. Aim 1 will develop next generation NLM technology and clinical workflow for rapid, comprehensive evaluation of prostate specimens in RP. These advances will enable a two person team (histotech/resident and pathologist) to perform comprehensive NLM of RP margins adjacent to the neurovascular bundles, faster and with much fewer personnel than NeuroSAFE. Aim 2 will perform a randomized controlled trial with patients undergoing robotic RP. The primary endpoints will be the rate of nerve-sparing RPs and rate of positive surgical margins in areas adjacent to the neurovascular bundles in a study group receiving intraoperative NLM margin assessment and standard-of-care postoperative FFPE histology versus a control group receiving standard-of-care FFPE postoperative histology. The secondary endpoints will be agreement between intraoperative NLM versus postoperative histology in the study arm and surgical times in the study arm versus control arm. Aim 3 will develop NLM technology and workflows that enable remote NLM evaluation which would increase access to pathologists with subspecialty expertise, streamline pathologist workflow, and facilitate adoption of intraoperative margin assessment in RP.

NIH Research Projects · FY 2025 · 2021-02

Project Abstract Sleep is essential for brain health, and neurodegenerative diseases are associated with substantial sleep disruptions. Disrupted sleep is now thought to not just be a symptom of neurodegeneration, but potentially to also contribute to the onset of the disease. Notably, Alzheimer’s disease pathology is associated with loss of EEG slow waves during non-rapid eye movement (NREM) sleep. Sleep is thought to be important for clearance of proteins such as amyloid-beta and tau from the brain into the cerebrospinal fluid (CSF), and the human brain exhibits waves of CSF flow during NREM sleep, suggesting that CSF flow during sleep may play a role in its effects on brain health. This proposal aims to understand the link between neural slow waves during sleep and CSF flow in healthy aging and in individuals at risk for Alzheimer’s disease. We hypothesize that neural activity can induce CSF flow through its effects on cerebral blood volume. We in turn predict that loss of neural slow waves during sleep in the aging brain may lead to loss of sleep-dependent CSF flow, and that this decline is associated with Alzheimer’s disease genetic risk factors. To test our hypothesis, we will use multimodal imaging to simultaneously measure neural activity, hemodynamics, and CSF flow. We will test the link between neural activity and CSF flow, and will identify whether the decline in sleep slow waves in older adults is associated with less CSF flow. We will further examine whether this process is more severely disrupted in healthy older adults with genetic risk for Alzheimer’s disease. Together, these studies will establish a biological mechanism for how altered sleep in aging leads to altered fluid flow dynamics, and this knowledge will form an essential foundation for the development of future biomarkers and interventions to evaluate and modulate CSF flow in the aging brain.

NIH Research Projects · FY 2025 · 2020-12

Model organisms have proven invaluable as more tractable systems to study fundamental principles of biology and by yielding breakthroughs that result from studying evolutionary innovations, for example, GFP, from jellyfish, or CRISPR, from bacteria. My goal is to leverage a new model system, developed during my postdoctoral work, that is uniquely positioned to provide insights of both types: Clytia hemisphaerica, a species of Mediterranean jellyfish. This proposal uses Clytia's distinctive features to identify precise mechanisms at the interface of neural development, neural regeneration, and systems neuroscience. First, Clytia are tiny (<1mm-1cm), transparent, and genetically tractable, making it possible to image and manipulate the activity of every neuron in the nervous system simultaneously, in vivo, using genetically encoded optical techniques. Further, Clytia numerically scale their nervous system at least an order of magnitude during their lifespan without disrupting system function, ending with more than ten thousand neurons. It is therefore also possible to observe continuous differentiation, migration, axon targeting, and functional activity simultaneously across the whole organism. Lastly, Clytia have poorly understood and powerful regenerative capabilities. These include the regeneration of large populations of genetically ablated neurons, with rapid recovery of the behaviors that those neurons control. These properties make Clytia an experimentally tractable platform to investigate: · Fundamental questions in systems neuroscience, including mechanisms underlying behaviors and behavior states, roles of neuromodulation, and approaches for studying system organization and function across scales. · Basic principles of neurodevelopment, particularly at the interface of development and function. · Mechanisms enabling regeneration and the seamless integration of new neurons into a functioning network. In Aim 1, I will characterize the molecular phenotypes of neurons and establish CRISPR-based knock-in to target effectors to specific subpopulations of cells. In Aim 2, I will use a coordinated behavior as my point of entry and develop and test models of underlying neural mechanisms. In Aim 3, I will examine how this behavioral system is robust to constant neurogenesis, and the mechanisms that enable its rapid recovery following genetic ablation of neurons. My vision is that, once the key foundational work has been completed and published, Clytia will become a widely used model system. This proposal serves as the first step, laying the foundation for my future independent program and for a Clytia community more broadly, and providing the essential training that I need to fill gaps in my knowledge, focusing on computational and single-cell RNA sequencing approaches.

NIH Research Projects · FY 2024 · 2020-12

PROJECT SUMMARY: Vimentin intermediate filaments in the cytoskeleton play an underappreciated physiological role in epithelial tissue development, repair, and tumor progression. In particular, vimentin knockout impairs mouse mammary gland development and regeneration in vivo, and disrupts 2D collective cell migration in vitro. Gain of vimentin is also associated with the epithelial-mesenchymal transition (EMT), where tightly-connected epithelial cells downregulate cell-cell adhesions and increase motility. For instance, “leader cells” at wound fronts can exhibit an elongated morphology with vimentin, while remaining partially connected to migratory followers. Indeed, a combination of vimentin and keratin 14 are associated with partial EMT states in genetically engineered mouse models of cancer. Nevertheless, the functional role of vimentin in multicellular migration, tractions, and coordination remains poorly understood, particularly in 3D matrix. Our long-term goal is to elucidate how the mechanobiology of the cytoskeleton regulates collective migration in organ formation, tissue repair, and disease. This goal requires addressing several fundamental questions: 1) How does vimentin affect directed migration and cellular deformability? 2) How does vimentin affect collective tractions? 3) How does vimentin affect cell-cell adhesions and collective migration? 4) How do vimentin-high and vimentin-low cells interact during collective migration? Based on these challenges, our objective is to elucidate the role of vimentin in collective migration through directed cell motility, multicellular tractions, as well as cell-cell coordination. Our approach will integrate several complementary technologies for precision measurement of collective cell migration and mechanobiology. MPI: Guo is an Early Stage Investigator with extensive expertise in the mechanics of soft and living materials, including vimentin networks and extracellular matrix. MPI: Wong is a New Investigator with extensive expertise in collective migration and EMT, particularly single cell tracking and analyses. Co-I: Goldman is a leader in the molecular biology of vimentin. This proposal is structured around 3 aims: elucidate how vimentin affects cell shape, migration and deformability of multicellular collectives in confinement (AIM 1), multicellular tractions in 3D matrix (AIM 2), and collective interactions of “mosaic” spheroids with heterogeneous vimentin expression (AIM 3). Overall, this work will reveal new fundamental insights into the role of vimentin in cell shape, motility, and mechanical integrity.

NIH Research Projects · FY 2025 · 2020-09

Music is a powerful memory cue that can reliably transport us to our past -- in effect, strengthening the associative bond between our present and our past. Modern neuroscientific investigations of episodic memory could benefit from the use of music due to its role as a potent memory cue. In this proposal, my goal is to understand how music shapes memory for past events and how these associations between events are represented in the brain. The proposed work builds on recent functional MRI studies that have used naturalistic experimental paradigms such as movie watching and recall to test and refine theories of memory that (previously) had only been tested under highly-controlled conditions. Here we explore how film scores shape memory for movies. In movies, film scores sometimes have recurrent structure where a particular theme song will be repeated throughout, potentially bringing back to mind information from previous events in which that theme song had been played. Based on prior work showing that retrieval of a memory strengthens that memory, we predict that the presence of recurrent musical structure in a film will trigger retrieval of previous events during movie-watching, which (in turn) will boost memory for these events on a later recall test. In our study, two groups of participants are recruited: The first group watches a version of a movie in its original form (with the film score intact) and the control group watches the same film with the entire film score removed (while preserving dialogue and ambient sounds). Participants from both groups are asked to return the following day to recall the entire movie in chronological order. By comparing neural activity between scenes containing music and previous scenes containing the same (or similar) music between the two groups, we expect to find evidence of neural reinstatement of past scenes in the music group; furthermore, we predict that higher levels of reinstatement during movie-viewing will be associated with better subsequent recall of the movie in the music group relative to the control group. This work will provide basic-science insight into how memory works in real-world contexts. It will also shed light on how musical cues can strengthen memories and improve recall; if the predicted cueing effects are observed, these techniques could be used in future work to boost performance in memory-impaired clinical populations.

NIH Research Projects · FY 2025 · 2020-08

PROJECT SUMMARY Immune-modulating therapies have been revolutionary treatments for several cancer types including melanoma, lung cancer, and renal cell carcinoma. However, most cancer patients do not respond to immunotherapies, and there remains a critical need to identify alternative approaches to treating these cancers. Endogenous retroviruses (ERVs) are genetic remnants of retroviral infection transmitted vertically through generations, whose transcription can result in type-I interferon activation, and/or presentation of ERV-associated antigens. Recent studies on ERVs in human cancer has shown that ERV expression can render cells immunogenic in a variety of cancer types including colorectal, breast cancer and melanoma. Therefore, characterization of how ERV expression is regulated in cancer will reveal opportunities to therapeutically de-repress ERVs and improve immunotherapy outcomes in low antigen tumors. In the F99-phase of this proposal, I will investigate the role of epigenetic factors regulating ERV expression and anti-tumor immunity in melanoma. Specifically, I will test melanoma tumors for ERV tetramer-positive CD8+ T cells, to determine the antigenicity of de-repressed melanoma ERVs. Furthermore, I will determine the mechanism by which type-I interferon response is induced in epigenetically-modified tumors, and determine the role of ERV MHC-I antigens through genetic knockouts of each component and evaluating tumor growth and immunogenicity. This work will establish the mechanism by which ERVs are regulated epigenetically, and establish a framework for investigating ERV regulation and its impact on the immune response to cancer. In the K00-phase of this proposal, I will harness my experience studying ERVs to investigate their utility in improving the immune response to antigen-low tumor types. Specifically, I will perform a CRISPR screen to identify druggable regulators of HERV expression in human cancer cell lines. I will then validate these candidate regulators by pharmacologically targeting them and testing the induction of type-I interferon and antigenic responses in these cells. Finally, I will evaluate whether ERV-specific CAR-T cells can react to ERV-induced cells in vivo. This proposed research will clarify the mechanisms by which ERVs are epigenetically regulated in cancer, and identify novel and actionable targets for re-expressing ERVs in difficult-to-treat tumor types. This will lead to improved immunotherapeutic strategies for treating cancers with poor patient outcomes.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT There is no shortage of innovative ideas for behavioral interventions to improve health care delivery in the United States. Health care delivery reform has the potential to dramatically improve the health and health care experience. This is especially true for the aging population who interact with the health care system more frequently and suffer some of the gravest health problems. However, too little is known about which interventions could be effective for improving the health of these individuals. This proposal seeks to identify, fund, and support low-cost, high-impact Stage I trials of behavioral interventions for mid-life and older people. The Abdul Latif Jameel Poverty Action Lab (J-PAL), founded in 2003, is a network of more than 165 affiliated professors at over 55 universities, united by their use of randomized evaluations to answer critical policy questions. J-PAL North America (NA) was launched at the Massachusetts Institute of Technology (MIT) in 2013 by Amy Finkelstein (MIT) and Lawrence Katz (Harvard) to apply the J-PAL approach to the region. J-PAL NA’s Health Care Delivery Initiative (HCDI) focuses on catalyzing high-quality randomized controlled trials (RCTs) to test how best to improve health care delivery. We propose to leverage this foundation to spur and support early-stage RCTs to test behavioral interventions to improve health care delivery and health outcomes for older adults. The MIT Roybal Center will support two Stage I pilots in its first year, and will select additional pilot projects in Years 2-5 through a rigorous peer review process. Our proposed Year 1 projects are: Project 1: Deferring Agency at End-of-Life: The Role of Information and Advance Directives. This project focuses on policies related to medical care and decision-making at end-of-life, a topic that is broadly relevant to the aging population. Project 2: Post-Acute Pain Management for Opioid-Tolerant Patients: A Randomized Controlled Trial. This project seeks to provide rigorous evidence on how to effectively manage pain in patients who are already on high doses of opioids and thus at risk of a host of negative outcomes, including overdose. Each of these projects will be supported by (i) the Management and Administrative Core, which will provide overall leadership and coordination for the Center and (ii) the Pilot Core. The Pilot Core will support project development activities to identify promising Stage I research projects, coordinate selection of the most promising pilots for funding, support funded pilots, and create a sustained behavioral intervention development program to improve health outcomes for older adults.

NIH Research Projects · FY 2024 · 2020-07

Abstract Blood vessels play a critical role in the circulatory system. The main function of blood vessels is transporting blood from the heart to the rest of the tissues and organs throughout the body and then bringing it back to the heart. The structures of blood vessels are crucial to their physiological functions. The intima consists of endothelial cells, which are intertwined with a polysaccharide intercellular matrix to form the lumen for blood transportation. In straight sections of a blood vessel, endothelial cells (ECs) typically align and elongate in the direction of blood flow. The media is the middle layer in the vessels, where the elastic fibers, polysaccharides, and vascular smooth muscle cells (SMCs) are mainly located. In particular, the circumferentially aligned SMCs in ring-like patterns control the constriction/dilation of the vessels, enabling modulation of hemodynamics. Tissue engineering has provided a promising strategy to repair and replace portions of tissues, where blood vessels are one of the most important yet challenging tissue to engineer. However, engineered blood vessels using conventional strategies based on scaffolds are usually produced using relatively sophisticated microfabrication procedures, and cannot be easily applied to vessels with complex architectures and/or small sizes. In comparison, the recent advances in the three-dimensional (3D) bioprinting technology have provided unprecedented flexibility in engineering blood vessels with high resolution, strong fidelity, and good complexity. Nevertheless, 3D bioprinting of structurally stable and functional vascular tissues has rarely been achieved. To this end, we propose to develop a unique bioprinting strategy, combining the digitally tunable microfluidic hollow fiber bioprinting method and the stretchable hydrogel-based bioink formulations, to generate structurally, mechanically, and functionally biomimetic non-branching macrovascular grafts of various sizes, shapes, and structures to significantly facilitate vascular transplantation.

NIH Research Projects · FY 2024 · 2020-06

PROJECT SUMMARY/ABSTRACT The objective of this project is to create an unobtrusive, wrist-worn, cuff-less blood pressure monitor for measurement and identification of nocturnal nondipping hypertension. The investigation includes extensive validation with state-of-the-art ambulatory blood pressure monitors at nighttime in presence of heterogeneous treatment paradigms. Cardiovascular disease (CVD) is one of the major causes of ailments worldwide. Hypertension alone affects one in three adults according to the World Health Organization. Therefore, monitoring blood pressure has become a critical part of healthcare as it is known to be linked to many CVDs. Traditionally, clinical practitioners have relied on the mercury-based (or digital equivalent) inflatable cuff-based sphygmomanometer. However, the nature of the device allows for only infrequent measurements and its somewhat invasive nature and associated discomfort prohibits additional nocturnal measurements. There is certainly a value to measuring blood pressure continuously in the natural context of the user’s environment, in particular during sleep, without being disturbed by the instrument. Our proposed technology can provide a wealth of information to physicians, help identify certain short-term dynamics/variations of blood pressure, and allow effective monitoring of response to medication, among other things. Nocturnal measurements provide additional prognostic value in identifying risk. Despite these benefits, no wearable, non-invasive device for continuous blood pressure monitoring exists on the market simply because none have been reliable enough to be considered clinical grade. This project aims to develop a robust and reliable blood pressure monitor in the form of a wrist-worn device that uses bio-impedance sensors, and for the first time, demonstrate clinical grade reliability. These sensors measure pulse wave velocity (PWV) along with several other derivatives for cardiovascular parameters including heart rate and blood volume changes in arteries, which correlate with the blood pressure. The system will incorporate clever hardware design to localize underlying vasculature and focus on arterial sites for enhanced accuracy. The device will include a motion sensor to take into account the user’s movements and motion artifacts, the contact quality, and reliability of the measurements. Advanced machine learning techniques, leveraging both general and personalized models, will be developed to convert bio-impedance measurements to blood pressure. This project aims to then validate the system and analytics in both a healthy patient cohort and a hypertensive cohort, learning the impact that nocturnal ‘nondipping’ hypertension and anti-hypertensive treatments have on PWV/other cardiovascular correlates and blood pressure estimates. After decades of relying on the inflatable cuff- based technique, this system could represent a significant change in how we measure blood pressure.

NIH Research Projects · FY 2025 · 2019-09

Project Summary Cancer cells have metabolic requirements that differ from most normal, non-proliferating cells. To proliferate, cancer cells must transform available nutrients into the varied array of macromolecules that are needed to build a new cell. Each cancer type is unique and will run a metabolic program that depends on the tissue-of- origin, genetic factors, and the local environment. How specific cancers integrate these cancer cell-intrinsic and extrinsic factors to rewire metabolism and support cancer progression is a major unanswered question. My laboratory's long-term goal is to understand how cancer cell metabolism is adapted to support tumor initiation and progression. The metabolic phenotypes of proliferating cells are typically interpreted with an emphasis on either energy generation or the crosstalk between signaling events and cell metabolism. This has led many to focus on how cancer genetics influences metabolic pathway use. We take a different approach that identifies limiting metabolic processes, considers how these are constrained by the extracellular environment, and defines how metabolic limitations are overcome within a physiological tissue context. Our work has provided insight into understanding how glucose metabolism affects cell proliferation. We found that production of nucleotides and oxidized biomass can be metabolic limitations of cell proliferation and tumor growth, and that both cancer cell-intrinsic and environmental factors determine how cells overcome these limitations. We have developed novel tools to study metabolism in various physiological contexts and uncovered metabolic differences between tumors and cancer cells in culture. We have demonstrated how environmental nutrients and cancer lineage can dictate how metabolism is used to support proliferation and determine sensitivity and resistance to drugs used in patients. Our work has charted new research directions for the field and contributed new ideas to exploit altered metabolism to help cancer patients. Using mass spectrometry to trace nutrient fate in cancer models, my laboratory generates hypotheses for how different cancers use metabolism to support cell proliferation and tumor growth. We test these hypotheses using a variety of biochemical and genetic approaches to define how nutrient availability, metabolic pathway regulation, and tissue context constrain how cells use available materials to proliferate. Our current interests include identifying which metabolic processes create bottlenecks for cell proliferation, determining how metabolism is different in different cancers, examining in detail the influence of tissue type, tumor genetics, and tumor microenvironment, and understanding how diet and whole body metabolism influence tumor metabolism and cancer progression. We aim to advance understanding of metabolic pathway biochemistry, its relationship to cancer and mammalian physiology, and identify how best to target metabolism for therapy.

NIH Research Projects · FY 2026 · 2019-08

Neuroscientific data contain information from an incredible diversity of species and modalities that are generated by a plethora of devices, and encapsulate the results of scientific thinking and decision making. The BRAIN Initiative has spearheaded a comprehensive informatics initiative to gather much of this neuroscientific data into standardized representations and to disseminate it through accessible platforms. DANDI - Distributed Archives for Neurophysiology Data Integration, is one such current effort to facilitate the aggregation and dissemination of neurophysiology research data using best practices and standards, and has grown to accommodate about 400TB of data across 100+ published datasets in slightly over 2 years. The archive supports a broad range of users with different levels of expertise by providing a spectrum from Web-based to programmatic mechanisms to access and upload data and helps improve the expertise through training of the scientific user base through tutorials and workshops. We expect future datasets to be larger and more multimodal, ranging in size from many TBs to PBs, with richer metadata. To support the next generation of neuroscience researchers and to support the scales of computation and storage that will become necessary, we must archive, preserve, and process this data in a scalable and accessible way that is meaningful to both neuroscience researchers and software developers. In Aim 1, we will integrate neurophysiology applications that scientists can easily use on large and diverse datasets to derive new insights and generate interactive figures, directly connecting the provenance claims to underlying data. In Aim 2, we will expand search functionality to query into the structure of individual data streams to enable more complex queries that enable more precise interrogation and advanced analysis of data and help answer more specific neuroscientific questions. We will improve search to span information within DANDI and to facilitate linking and integration of DANDI data with related data available in other BRAIN Initiative archives. In Aim 3, we will improve interoperability of data in DANDI with other neurophysiology software tools, platforms, and applications, thereby strengthening the ecosystem of neurophysiology research. Community engagement and data reuse will be further enhanced through yearly workshops aimed at improving the quality of data and metadata and training users to use DANDI tools and data. Overall, we will address the growing data management and dissemination needs of the neurophysiology community through a scalable, robust, interoperable, and standards-based neurophysiology archive that provides an easy to use graphical and interactive interface as well as computation services close to large datasets that can be accessed simply with a Web browser. We will provide a platform for seamlessly integrating with and enhancing existing research workflows. We aim to support scientific inquiry and collaboration, reduce redundancy in computation, and preserve and present information according to FAIR (Findable, Accessible, Interoperable, Reusable) principles.

NIH Research Projects · FY 2026 · 2019-08

PROJECT SUMMARY Biomolecular condensates are compartments that form within cells through the self-assembly of proteins, RNAs, etc. These condensates play a critical role in the functioning of cells as they help to concentrate biomolecules and facilitate biochemical reactions. Recent studies have shown that they are also essential for transcriptional regulation. However, there are still many unanswered questions about how these condensates form, interact with each other, and how they organize chromatin and regulate transcription. One of the challenges in addressing these questions is the lack of suitable experimental techniques for quantitatively studying the spatiotemporal complexity and heterogeneity of transcriptional condensates. We propose to characterize transcriptional condensates in the context of chromatin using in silico approaches. (i) By introducing many-body potentials represented using graph neural networks and novel parameterization schemes, we will introduce the next generation of residue-level coarse-grained (CG) models for proteins, RNAs, and DNAs. These models will outperform existing ones that often lack the accuracy needed for de novo predictions, enabling their application from generating testable hypotheses. (ii) Using CG models that balance computational efficiency and chemical accuracy, we will reconstruct in vivo chromatin organization at near-atomistic resolution with a multiscale approach. We will evaluate the stability of these near-atomistic models and the contribution of physicochemical interactions in stabilizing chromatin. In addition, we will quantify the role of transcriptional condensates in chromatin looping and accelerating transcription factor target search using reconstructed in vivo chromatin structures. (iii) We will also decode the molecular grammar of protein-protein interactions that dictate condensate stability using evolutionary sequence analysis and physical simulations. We will use this information to design small molecules for condensate modulation. Overall, the goal of our research is to provide critical evaluations of hypotheses on the role of transient, collective phenomena arising from weak, multivalent interactions in essential cellular processes and suggest novel therapeutic strategies for their modulation.

NIH Research Projects · FY 2024 · 2019-07

PROJECT SUMMARY Our relationship with the visual world is heavily dependent on our ability to receive and process light coming into the eye. A portion of that light-derived information is needed for many non-image-forming visual functions like programming our biological clocks, controlling eye movement, and influencing mood. A major region of the brain which receives this non-image-forming visual information is located in the ventral thalamus and known as the ventral lateral geniculate nucleus (vLGN). Although the vLGN has connections with downstream regions involved in important functions, including visuomotor function, circadian photoentrainment, and vestibular function, it has not been well-studied. One reason for this significant gap in knowledge is that very little is known about funda- mental characteristics of vLGN, particularly with respect to its cellular and molecular landscape, thus making it technically challenging to develop tools to answer outstanding questions about its role in visual information pro- cessing. This study is motivated by the need to characterize the molecular architecture of vLGN, and to identify the neurons which form connections with retinal fibers. This project directly aligns with the BRAIN Initiative and the NIH's Blueprint Program by expanding fundamental understanding of neuroscience using innovative re- search methods to characterize cell types in the nervous system and mapping connected neurons in the brain. The results of this work will significantly impact the field and generate a deeper understanding of a visual region of the brain which receives, processes, and transmits non-image-forming visual information.

NIH Research Projects · FY 2026 · 2019-07

An immunosuppressive or immune excluded tumor microenvironment (TME) plays a key role in limiting the response of many tumor types to immunotherapy. One attractive strategy to accomplish increased lymphocyte infiltration in tumors is the use of cytokines, which can directly impact multiple immune pathways and reprogram the TME to enable a robust immune response against cancer cells. Unfortunately, despite this obvious potential, many cytokines have been limited clinically due to toxicity concerns. Rational drug delivery strategies that can rescue the therapeutic potential of cytokines could act as an important step in our ability to carefully manipulate the anti-tumor immune response in the TME and open the door for more effective immunotherapies. The layer- by-layer (LbL) approach allows us to modify NP surface properties via electrostatically absorbed polymeric coatings that promote specific cancer cell association and influence intracellular trafficking. In the first funding period of this grant, we employed this strategy to target proinflammatory cytokines such as interleukin-12 (IL-12) directly to OC cell surfaces. This therapeutic approach elicited robust CD8+ T-cell and NK cell infiltration into the TME, and when coupled with combination checkpoint therapies (CTLA-4 and PD-1), this treatment strategy yielded complete remission in a metastatic ovarian tumor model and promoted effective immune memory. The successful improvement of IL-12 localization and retention within the TME demonstrated the potential of IL-12 as a monotherapy; however, in many cases, IL-12 alone is insufficient to promote curative responses and often a combination treatment is needed. Messenger RNA (mRNA) provides a viable alternative approach to deliver combinations of cytokines and engineered immunomodulatory therapeutics, as mRNA readily enables combinations due to facile exchange of different nucleic acids and potentially lower systemic exposure of high bolus doses due to prolonged and targeted expression. The rapid development of mRNA constructs and their facile encapsulation into lipid nanoparticles (LNPs) allows for the rapid screening of therapeutic constructs in a high throughput manner, whereas in contrast the use of an engineered protein therapy requires extensive protein production optimization and purification steps. This mRNA-based approach will thus allow us to facilitate clinical translation of cytokine therapies while introducing targeted mRNA production as a means of addressing systemic toxicity. In this renewal, we seek to maintain or improve the extended duration of cytokine bioavailability gained in current work, while facilitating combination cytokine therapies by developing layered lipid nanoparticles (LLNPs) for the targeted delivery of cytokine-encoding mRNA to OC tissues. We will develop, optimize, characterize and test LLNPs encoding for IL-12 cytokine, IL-12 fused to an anti-CD45 Ab for targeting leukocytes in the TME, and (3) IL-12 fused to a GPI anchor. We will test these treatments in a range of highly metastatic ovarian cancer models and leverage the platform to incorporate combination cytokines IL12 and IL15, as well as combinations with checkpoint inhibitors in orthotopic syngeneic animal models. 1

NIH Research Projects · FY 2026 · 2019-01

PROJECT SUMMARY. To find an object in a complex scene, we use feature-based attention to guide our search, typically in conjunction with spatial attention and targeting eye movements. When searching for our keys on a table, for example, the features of the keys are used as an attentional template that guides the eyes to the various objects sharing features with keys until the keys are found. Work from our own and other labs has found that objects with attended features or attended locations are processed more efficiently in visual cortex, while the processing of unattended, distracting objects is suppressed. To design an effective neural prosthesis or to treat people with sensory or attentional impairments, we need a better mechanistic understanding of these attentional mechanisms at the systems level. The interconnected structures important for the control of attention have many common features. At the surface level, these common features suggest there may be little difference in their functions. However, our results show surprising specificity instead. We find that the ventral pre-arcuate area (VPA) and the frontal eye fields (FEF) in prefrontal cortex (PFC) have different functions in visual search. Specifically, VPA appears to mediate the selection of likely targets based on their features, and FEF directs spatial attention and gaze to those possible targets until the object of the search, the target, is found. We hypothesize that VPA and FEF work together as an interconnected system for guiding gaze to objects we are searching for, and that they also provide attentional feedback to the occipital and temporal cortex, including the mid-superior temporal sulcus (mid-STS) region. This feedback biases visual processing in favor of attended target features. In Aim 1, we will test whether feedback from FEF to visual cortex and VPA is specific for attended locations, as we propose, or whether the feedback conveys priority values computed from both attended features and locations. In Aim 2, we will use electrical stimulation coupled with functional magnetic resonance imaging (fMRI) to finely map the connectome of the mid-STS region, which receives projections from VPA and has been recently proposed as an important component of the system for the top-down control of attention. The published connectome will provide a test of anatomical mapping principles that we recently discovered in PFC, and it will be a valuable resource for the neuroscience community. In Aim 3, we will use neurophysiological recordings in VPA, FEF, the mid-STS, and other structures proposed to be important for attention, coupled with causal methods such as optogenetics and muscimol injections to test our hypotheses about the roles of the different components. One of the key innovations of this project is that we will map the connectivity of the recorded sites with electrical stimulation and fMRI so that we can target our recordings to the specific neuronal groups in different structures that are interconnected with one another. In total, we expect these studies to give us the best account so far of how the interactions among multiple brain structures leads to effective visual processing during attention to object features.

NIH Research Projects · FY 2026 · 2018-08

Project Summary - Despite decades of research into Alzheimer's disease (AD), disease-modifying treatments for AD remain elusive. This is significantly due to challenges in understanding the molecular and structural basis of AD. One of the two hallmarks of AD is the neurofibrillary tangles formed by the intrinsically disordered microtubule (MT)-associated protein tau. Spreading of tau filaments in the brain is the basis of neuropathological staging of AD. AD tau is hyperphosphorylated, truncated, and decorated with other posttranslational modifications (PTMs). However, how these PTMs cause tau to dissociate from MTs and misfold into -sheet amyloids, and how tau crosses the lipid membrane to spread its pathology, is not known. AD paired helical filament (PHF) tau fibrils have a C-shaped -sheet core that encompasses part of the MT-binding repeats. But the majority of the protein, which contains most of the disease-relevant PTMs, is too disordered to be seen in cryo-electron microscopy data. Here we propose to employ solid-state NMR (ssNMR) spectroscopy, electron microscopy, mouse neuron toxicity assays, and other biochemical approaches to understand the molecular structures and dynamics of AD tau filaments, membrane-bound tau, and MT-bound tau. We hypothesize that specific charge-charge interactions underlie the varying conformations, dynamics and properties of tau when self-aggregated and when bound to its cellular partners. In the last four years, we demonstrated the feasibility of applying ssNMR to study the structures and dynamics of full-length tau fibrils formed in vitro and seeded by AD PHF tau. We will now apply this expertise to answer three questions. In Aim 1, we will investigate how phosphorylation and truncation cause AD PHF tau by determining the structures of phosphorylated tau (p-tau) fibrilized without anionic cofactors; searching for minimum constructs that replicate the AD PHF tau structure and properties; and characterizing the dynamic structures of the semi-mobile proline-rich region of tau. In Aim 2, we will investigate tau interactions with lipid membranes by measuring the conformation, dynamics and membrane insertion of monomeric tau bound to small and large unilamellar vesicles. We will determine the structures of membrane-induced tau aggregates, and probe how phosphorylation and truncation affect the structure and dynamics of membrane-bound tau. These experiments should shine light on how lipid membranes nucleate tau aggregates and how aggregated tau crosses the membrane. In Aim 3, we will investigate the structures of MT-bound tau as a function of phosphorylation, and probe how arginine-phosphate interactions in the R' domain affect tau binding to MTs. A joint study of the fibrillar, membrane-bound and MT-bound tau is crucial for understanding how tau converts from its intrinsically disordered structure to an aggregated structure that propagates in a prion-like manner. This understanding should inform the future design of drugs that interfere with this process.

NIH Research Projects · FY 2026 · 2018-05

The combination of metal ions with proteins offers unique chemical reactivities that are at the heart of many of Nature’s most important and amazing chemical transformations. For example, metalloenzymes catalyze the reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA biosynthesis. They biosynthesize anticancer and antiviral compounds and vitamins that have unusual scaffolds. Our lab employs structural methods to interrogate how metalloenzymes are able to perform this incredible chemistry. We seek to understand how the architecture of metalloenzymes allows for radical species to be controlled – i.e. turned off, turned on and harnessed – to enable the reaction at hand. We also strive to understand how proteins are designed to enable long-range electron transfer without protein damage or radical loss. In this proposal, we describe structural studies of our metalloenzyme model systems, including class Ia (diiron-dependent) and class III (glycyl radical-dependent) ribonucleotide reductases that allow us to interrogate the molecular basis of radical-based chemistry. We also describe efforts to understand how protein scaffolds facilitate organometallic chemistry. These studies leverage both our expertise in working with O2-sensitive metalloenzymes and in cryogenic-electron microscopy (cryo-EM). Although we will continue to employ X-ray crystallography, cryo-EM is proving to be a game-changer for many of our metalloenzyme systems. In particular, the resolution revolution of cryo-EM provides us with the means to obtain long-awaited structures of both large and transient metalloprotein complexes and to determine structures of metalloenzymes in functionally-essential conformational states that were previously unattainable by crystallography. The results of our structural studies will enable structure-based design of novel antibiotics targeting, for example, microbial ribonucleotide reductases. They will aid our understanding of human ribonucleotide reductase, a major cancer drug target. These structural data will also guide efforts to exploit radical enzymes for the production of medically important compounds with unusual scaffolds.

NIH Research Projects · FY 2024 · 2017-09

PROJECT SUMMARY The primary objective of this renewal application is to elucidate how the metal-sequestering host-defense protein calprotectin (CP) modulates the physiology and interactions of Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) using models that consider environmental cues relevant to infection. Metal ions are essential nutrients for all organisms, and pathogens must acquire these nutrients from the host to replicate and cause infection. In response to pathogen invasion, the human innate immune system enacts a metal- withholding response to limit the bioavailability of transition metal nutrients including manganese (Mn), iron (Fe), and zinc (Zn). This host response includes the deployment of CP and other metal-sequestering proteins at sites of infection. We discovered that CP withholds Fe(II) from and induces Fe-starvation responses by Pa and Sa, two bacterial pathogens that cause chronic polymicrobial infections in diverse patient populations, including lung infections in individuals with cystic fibrosis (CF). This hereditary disease predisposes individuals to life-long pulmonary infections, marked by debilitating exacerbations that reduce lung function. Notably, the CF lung becomes increasingly acidic and hypoxic as disease progresses, and Fe(II) becomes a predominant form of bioavailable Fe in this environment. Nevertheless, few studies have addressed how acidic pH and hypoxia impact microbial Fe homeostasis and the host metal-withholding response. We hypothesize that the Fe(II)-sequestering activity of CP has profound consequences on the physiology and virulence potential of Pa and Sa in diverse environmental milieus relevant to infection. We further propose that interactions between CP and these pathogens depend on environmental cues that vary both temporally and spatially at infection foci. In Aim 1, we will evaluate how mildly-acidic pH affects the metal-sequestration profile of CP as well as the physiology of Pa and Sa and their responses to CP and metal starvation. In Aim 2, we will examine how hypoxia affects the responses of Pa and Sa to metal starvation and CP. In Aim 3, we will extend these investigations to more complex in vitro models that incorporate relevant aspects of infection including co- cultures, biofilms, and additional metal-sequestering host-defense factors such as lactoferrin and S100A12. These investigations will enable future studies that address how CP and Fe drive the progression of CF lung infections and may guide the design and development of novel diagnostic, preventative, and therapeutic approaches to treat bacterial infections.

NIH Research Projects · FY 2026 · 2017-09

ABSTRACT: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with few effective treatments. The poor efficacy of current therapies including immune checkpoint inhibitors (ICIs) is partly attributed to the characteristic fibroinflammatory desmoplastic tumor microenvironment. Thus, strategies that overcome these stromal barriers have the potential to profoundly improve therapeutic outcomes in PDA. Towards this end, epigenetic therapies that broadly rewire cellular gene expression programs represent a promising approach for targeting PDA stromal barriers. Preliminary studies with inhibitors of Bromodomain and Extra-Terminal domain (BET) proteins, whose recognition of enhancer and super-enhancers drive cell-specific function, reveal a potent loss of immunosuppressive programs within multiple stromal cell populations as well as tumor cells. In addition, the clinical BET inhibitor OTX-015 (OTX) synergized with otherwise ineffective αPD-L1 immune checkpoint inhibition to promote intra-tumoral cytotoxic T cell activation and decrease PDA tumor burden. However, OTX negatively impacts T cell priming in secondary lymphoid tissue and long-term treatment is limited by systemic toxicities. To overcome these limitations, this proposal will develop bottlebrush polymer prodrug (BPDs) to selectively deliver drug cargoes to PDA tumors. BPDs are small, cylindrical macromolecules with multiple conjugated drugs within their cores. This unique architecture enables improved tissue penetration and predictable properties independent of drug composition, while molecular linkers facilitate highly selective drug release in target tissues. In Aim 1, the ability of OTX-BPD conjugates incorporating cleavable linkers to selectively deliver drugs to tumors will be evaluated in clinically relevant PDA mouse models. Subsequently, the ability of lead OTX-BPDs to synergize with αPD-L1 will be determined in both short-term intervention and long-term survival studies. The therapeutic utility of conjugating multiple drugs to BPDs will be determined in Aim 2. Specifically, chemotherapeutics found to synergize with OTX as free drugs will be conjugated to OTX-BPDs, and the abilities of these multidrug laden BPDs to reduce tumor growth and enhance anti-tumor immunity in combination with αPD-L1 will be determined in PDA mouse models. In parallel, the therapeutic benefits of actively targeting these multidrug-laden BPDs by conjugating to antibodies that recognize PDA cancer cell proteins will be assessed. In addition, the optimal ratio of drugs conjugated to individual BPDs to achieve maximal efficacy will be established, which will be distinct from free drugs. In combination, the development of targeted multidrug-laden BPDs will promote rational combination therapies that leverage both epigenetic- and chemo-therapies to potentiate immune checkpoint inhibitors. In the third aim, BPD reporters for drug-induced apoptosis and cytotoxic T cell activity will be developed by incorporating metal-free MRI organic radical contrast agents. These reporter BPDs will be used to validate the method-of-action of combination therapies developed in Aim 1 and Aim 2. Importantly, the ability of such BPDs to rapidly report on treatment efficacies has the potential to inform subsequent treatment decisions.

NIH Research Projects · FY 2025 · 2017-09

N-Nitrosamines are a family of hazardous chemicals that are among the most mutagenic chemicals known, and many are potently carcinogenic in animal models. People living in Wilmington, MA, are concerned about N- nitrosamines because there are over 20 million gallons of N-nitrosamine- contaminated waste at the nearby Olin Chemical Superfund Site, contaminating the environment and rendering their well water undrinkable. The Passamaquoddy Tribe is also concerned about N- nitrosamines, because their water contains high levels of organic material and the use of chloramine is known to create N-nitrosodimethylamine. Specific Aim 1 is to make and measure. Innovative, light- based chemical sensors that exploit smartphones will be created and used for Citizen Science to gain information that will help to inform cleanup by the Environmental Protection Agency (EPA). Innovative, high-throughput ‘animate sensors,’ based on cell-microarray technology, will be created and used to test the consequences of N-nitrosamines on health-related impacts known to be associated with cancer risk. To understand disease more deeply, a genetically engineered “canary in the coal mine” mouse model will be used to reveal the potential for long-term low-dose exposure to cause mutations and deleterious biological responses. Specific Aim 2 is to protect human health via prediction and prevention of disease. By integrating multi-omics data (fueled by the Data Management and Analysis Core [DMAC]), mechanistic knowledge will propel the development of predictive biomarkers that can be used to develop methods to prevent disease. The potential for probiotics to suppress N-nitrosamine- induced cancer will be studied. In addition, novel devices will be created to destroy N-nitrosamines via electrochemical and biochemical destruction. Importantly, risk evaluation depends on knowledge from both engineers and biologists. The DMAC will thus form a critical integrating role by merging transdisciplinary data streams to evaluate risk for specific water samples. Specific Aim 3 is to maximize societal impact via integration within the MIT SRP and with partners outside of MIT. Partnering with the community will allow collection of environmental data that will inform risk. Via bidirectional communication, community members will also benefit from novel, hands-on teaching kits, while MIT SRP members will benefit from learning about community perspectives and concerns. Dissemination of MIT SRP knowledge and technologies will also be achieved via continued strengthening of relationships between the MIT SRP and local, state, Tribal and governmental agents. All of this work will be made possible by careful coordination and formalization of translation opportunities (made possible by the Administrative Core), and by continuous improvement of training opportunities that not only fuel the research (made possible by the Research Experience and Training Coordination Core [RETCC]), but also help to ensure that trainees go on to contribute as responsible leaders who are able to leverage cross-disciplinary research in order to have a powerful impact on public health.

NIH Research Projects · FY 2024 · 2017-09

Project Summary: The long-term goal of our laboratory is to understand how specific protein kinase signaling pathways function together with phosphoserine/threonine-binding domains and RNA binding proteins (RNA- BPs) to regulate tumor development after exposure to inflammation and genotoxic stress. We are particularly interested in understanding how these pathways can be manipulated to enhance cancer prevention, as well as to improve the response of any tumors that do form to conventional anti-cancer agents. In addition to the two canonical DNA damage response pathways that cells use to respond to DNA damage, the ATR-Chk1 pathway, and the ATM-Chk2 pathway, we recently identified a third DNA damage response pathway mediated by p38MAPK and MAPKAP Kinase-2 (MK2) that is absolutely essential for p53-defective tumor cells to survive after genotoxic stress. Importantly, the MK2 pathway is dispensable in cells with intact p53 function, making it an ideal target for specifically impairing the ability of cells undergoing cancer transformation to survive additional DNA damage. Unlike the ATR-Chk1 and ATM-Chk2 pathways that are dedicated to responding solely to signals from DNA damage, the p38 MAPK-MK2 pathway is a global stress-response pathway activated by multiple types of cellular stress, and plays a critical role in cytokine production during inflammation and early tumor development. Thus, we believe that the p38MAPK-MK2 pathway plays a particularly novel role during oncogenesis following genotoxic stress by integrating DNA damage response pathways within the damaged cells with inflammation and cytokine signaling arising in the adjacent stromal microenvironment. Importantly, both the DNA damage response function, and the cytokine production function of MK2, as well as many of the activities controlled by ATM-Chk2 and ATR-Chk1, appear to be mediated, in large part, by the action of RNA-BPs, which control gene expression at the post-transcriptional level. Finally, we and others have observed that certain xenobiotics appear to cause cell injury and death not through DNA damage, but instead through a distinct RNA damage response that has been very poorly characterized to date. In this proposal we (1) investigate the role of MK2 signaling in both the epithelial compartment and the inflammatory microenvironment in murine models of genotoxic stress-induced cancer development; (2) elucidate the emerging roles of RNA-binding proteins as key mediators of the cellular response to DNA damage; and (3) explore a poorly understood RNA damage response that induces profound apoptosis in a wide variety of epithelial tissues. The flexibility afforded by R-35 mechanism allows us to pursue these questions using a wide variety of combined experimental and computational approaches. The resulting mechanistic models are then tested in vivo using murine models of environmental stress-induced cancer and by querying human patient derived datasets, in order to achieve a transformational impact in the environmental health sciences.

NIH Research Projects · FY 2026 · 2017-09

Project Summary/Abstract The proteome is a quantitative output of a genome and the ultimate effector of cellular functions. Yet remarkably little is known about the logic behind proteome construction. The goal of my research program is to understand the evolutionary driving forces and the molecular processes that shape protein levels in living cells. Using bacterial model systems, my lab takes a holistic approach by developing quantitative technologies to measure, manipulate, and model the expression stoichiometry of co-regulated proteins and the effect of imbalanced production on bacterial growth, survival, and colonization. This current grant seeks to answer several fundamental questions regarding the physiology and regulation of protein stoichiometry. At the physiological level, we use molecular and theoretical methods to determine 1) the optimality of protein production rates for every gene and the cellular responses to non-optimal levels, 2) the precise ratios of protein production upon sudden environmental changes and the consequences of imbalanced production, and 3) the theoretical basis for the stoichiometry of proteins that function together. These studies will help elucidate new principles for building a proteome and provide a new way of thinking about protein imbalance in disease. To understand the regulatory principles of gene expression behind this stoichiometric protein production, we use quantitative and massively parallel assays to determine 1) the sequence determinant of Rho- dependent transcription termination in Bacillus subtilis, whose RNAP polymerases outpace ribosomes (`runaway transcription'), 2) the molecular basis of runaway transcription, 3) a predictive model for the efficiency of Rho-independent transcription termination, and 4) the landscape of translational control for bacteriophage mRNAs. Our studies in these areas will help establish a critically needed framework for predicting gene expression from genomic sequences and advance fundamental knowledge of bacterial gene regulation. We anticipate that our mechanistic dissection, coupled with systems-level inquiry into proteome composition, will make bacterial model organisms the first system for which we have a quantitative understanding of the interplay between genome, proteome, and fitness.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY The overarching goal of this renewal application is to elucidate the molecular basis for how human calprotectin (CP, S100A8/S100A9 oligomer) functions in the metal-withholding innate immune response, and to evaluate its impact on the physiology of uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections in humans. Transition metals are essential nutrients for all organisms, and the availability of these nutrients plays a critical role during microbial infection. Consequently, the human innate immune system launches a metal-withholding response and deploys metal-sequestering host-defense proteins into the extracellular space to limit metal availability and hinder pathogen growth. CP is an abundant and functionally versatile metal-withholding protein; it sequesters multiple metal nutrients including Mn(II), Fe(II), Ni(II) and Zn(II). Recent studies by our laboratory and others provide compelling evidence that the molecular speciation of extracellular CP is a heterogenous ensemble of different species that arises from different metal-bound forms as well as oxidative post-translational modifications. We hypothesize that this complex molecular speciation of CP, including the occurrence of methionine oxidation and disulfide bonding, has profound consequences for its extracellular function and lifetime. Recent studies by our laboratory and others also demonstrate that CP is a Cu-withholding protein. We hypothesize that CP sequesters both Cu(II) and Cu(I) and that this function impacts the physiology and metal homeostasis in diverse bacterial pathogens including UPEC. In Aim 1, we will examine disulfide bond formation within and between CP heterodimers, the biophysical properties of these disulfide-linked species, and their ability to sequester metals from bacterial pathogens. In Aim 2, we will evaluate the Cu(II/I)-binding properties of CP and the consequences of multi- metal sequestration by CP on UPEC as a case study. We expect that these investigations will advance the molecular model for how CP contributes to the metal-sequestering innate immune response, underscore the importance of considering CP species that result from oxidative posttranslational modification, and elucidate the molecular basis for Cu withholding by CP. Moreover, we expect that our studies of the interplay of CP and UPEC will provide new insight into how the host and pathogen compete for Cu and other nutrient metals. We further expect that the outcomes of this initiative may guide the design and development of novel diagnostic, preventative and therapeutic approaches for microbial infections and other pathologies such as inflammatory diseases that involve CP.

NIH Research Projects · FY 2026 · 2017-08

Project Summary/Abstract The proposed research focuses on the application and development of magic angle spinning (MAS) NMR as a tool for structural investigations of amyloid peptides and proteins. The research covers four major topics. A. Structure of Amyloid Peptides and Proteins (1) Aβ1-42 and Aβ1-40: Using 1H detected and DNP magic angle spinning (MAS) and cryoEM we plan the following experiments on Aβ: (a) a determination of the structure of Aβ1-40; (b) the structure of the pathologically important plaque seeded Aβ1-42; (c) the structure of the N-terminal tail and the binding of antibody Aducanumab to the tail; (d) and the structure of mutants for Aβ1-42. (2) Beta-2-microglobulin (β2m) and DeltaN6-β2m: We plan to determine the structure of the 93 AA β2m-DeltaN6 variant of the dialysis related amyloidosis (DRA) protein. In addition, DeltaN6 is thought to be the catalyst that seeds β2m plaques in-vivo. We therefore also intend to study mixtures of β2m and DeltaN6. (3) Amyloid polymorphism: We intend to determine the structure of the three polymorphs of the amyloidiogenic peptide GNNQQNY from Sup35. These are present in a consistent 1:1:1 population and will provide the first study of the manner in which defined structural changes alter 13C and 15N shifts. 1H, 13C and 17O NMR will be used to characterize the structure around the H2O molecules in the GNNQQNY lattice. B. NMR methods None of the above structural studies would be possible absent NMR methods to assign spectra, measure distances and torsion angles, to enhance signal intensities, etc. We therefore plan to continue the development of the methods essential for these structural investigations. (a) We plan to continue these experiments by initially preparing U-17O/13C/15N GNNQQNY to further develop the spectroscopy and then to apply it to spectroscopy of Aβ1-42 and Aβ1-40. The experiments will employ 1H detection and DNP in order to optimize the sensitivity. PAR and PAIN have been essential to MAS NMR structure determinations but they are semiquantitative methods to measure 13C-13C and 13C-15N distances. Using SPINEVOLUTION software and experiments on model systems, we plan to develop these approaches into quantitative distance measurement techniques.