Massachusetts Institute Of Technology

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Cardiogenic shock, a devastating outcome of decompensated heart disease, has both increased in incidence and maintained remarkably high mortality rates near 50%. Mechanical circulatory support is emerging with the unique ability to decouple cardiac supply and demand to sustain end-organ perfusion while reducing cardiac work. However, methods to guide device selection and titration are limited. One aspect of this which remains understudied is the importance of interactions between the left and right ventricles and their power over patient tolerance to device support. This is particularly critical as >40% of patients with left-sided support have been reported to experience right heart failure after device initiation, making this a major limitation to clinical utility. Thereby, this research employs a mechanistic approach to define the nature of right-left ventricular coupling in health and cardiogenic shock and its governance of mechanical circulatory support outcomes. We will employ a novel porcine model of graded left and right ventricular collapse to determine metrics critical to right and left heart adaptability individually, and those which contextualize the two to assess ventricular coupling. We will use this understanding to assess the biventricular response to two forms of clinical mechanical support. First, we will test a percutaneous left ventricular assist device, which employs a mechanism of continuously unloading the left ventricle to increase forward flow from the heart. The second technology will be veno-arterial extracorporeal membrane oxygenation support, which increases perfusion through venous withdrawal and retrograde return into the systemic circulation; this mechanism is particularly notable for unloading and reducing stress on the right heart and pulmonary circulation. Controlled stimulus provided by each technology will allow for assessment of the dynamic response according to ventricular coupling state. This project implements mechanistic analysis of the ability of each ventricle to respond to stress, as well as dynamic assessment (rather than classic static metrics) of ventricular-ventricular interactions (including serial interactions through the pulmonary circulation, parallel interactions through shared intracardiac structures, and synchronous interactions in time). In doing so, this work will provide insight into the governing factors over the response to mechanical support, allowing for improvements in device selection and titration, and ultimately improve outcomes from the devasting consequences of cardiogenic shock. I am excited to conduct this work with MIT Professor Elazer Edelman and to couple the research with a rigorous training plan which will allow me to grow as a scientist, communicator, and teacher. I am blessed to be at an institution and laboratory which provides all resources to complete this novel work and creates a research environment and training program that supports me in advancing towards independent translational research.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Oxidative stress is a byproduct of energy production necessary for all living organisms and caused by unregulated reactive oxygen/nitrogen/carbonyl species (ROS/RNS/RCS) among others. Nature has evolved the oxidative stress response (OSR) as a key component of metabolism that maintains cellular homeostasis by detoxifying and neutralizing aberrant reactive molecules. Spatiotemporally control of OSR is achieved through compartmentalization and redundancies that are coupled to create a redox balance to promote survival. Unbalanced OSR due to a defective or overactive capacity to resolve oxidative damage is associated with various human diseases. For example, chronic OSR is a hallmark of obesity, a global epidemic as well as a major risk factor for developing cardiovascular diseases, metabolic syndrome, and cancer. To better understand obesity, it is paramount that we elucidate the coordination of the OSR metabolon, defined here as the sequential antioxidant enzymes, biochemical reactions, and cellular compartments that maintain redox homeostasis. The proper regulation of and adaptive changes by OSR require rapid signaling taking place in the seconds-to- minute timeframe. Such dynamics must therefore require fast regulatory networks such as protein post- translational modifications (PTMs). Phosphorylation of serine (S) (~90%), threonine (T) (~9%), and tyrosine (Y) (~0.1-1%) residues are one of the many ways cells regulate pathways that maximize survival. Initial evaluation of the published phosphoproteome stratified by enzyme classification and pathway enrichment analysis indicates that, despite low intracellular stoichiometry, pY are enriched on antioxidant enzymes. However, the majority of pY sites on antioxidant enzymes are not functionally characterized. My overarching goal in this proposal is to gain network level insight into the pY directed regulation of antioxidant enzymes and the resulting dynamics of dysregulated OSR. I hypothesize that obesity-driven pY on multiple antioxidant enzymes modulates their catalytic activity to produce systemic changes in OSR. I will test this hypothesis by employing proteomics, metabolomics, structural analysis, and computational modeling. During the mentored phase of this application, I will predict the functional role of previously uncharacterized pY, validate predictions using in vivo as well as in vitro enzyme kinetic assays, and demonstrate pY-driven OSR dysregulation in an in vivo high-fat diet (HFD)- induced obesity mouse model. Through these interdisciplinary approaches, I aim to define systems of pY- modified enzymes that “tune” metabolic response to HFD, and evaluate differential regulation of OSR in a sex specific manner. Additionally, I will determine how altered dietary serine, glycine, or addition of small molecule antioxidants ameliorate HFD phenotypes, and the sex specific responses in the OSR metabolon that may be therapeutically relevant. This proposal and the outlined training plan will equip me with the technical skills, scientific knowledge, and professional training that will serve as the foundation to launch my research focused on OSR regulation as an independent investigator.

NIH Research Projects · FY 2026 · 2023-09

ABSTRACT In the United States, cancer is the second leading cause of death, and it is projected that 39.5% of all US adults will be diagnosed with cancer in their lifetimes. Carcinomas comprise up to 90% of all US cancer cases. Chemotherapeutics and antibody immune checkpoint inhibitors (ICIs) are promising treatments for these cancers but are not effective in all cases and exact a large toll on the quality of life of patients due to off-target toxicity. The goal of this proposal is to develop a peptide-based hydrogel therapeutic platform for the local delivery of chemotherapeutics and ICIs to maximize treatment efficacy and mitigate systemic toxicity. Boronic acid-containing small molecule drugs (BACSMs) are a growing class of chemotherapeutics for the treatment of cancer. Bortezomib is an FDA-approved BACSM for the treatment of multiple myeloma and causes immunogenic cancer cell death to help the body develop an anti-cancer immune response. There is interest to expand the use of bortezomib to solid tumors but these efforts face challenges due to the inability to maintain high local concentrations in the tumor without systemic toxicity. Multidomain peptide (MDP) hydrogels are self-adjuvanting materials that have been investigated for cancer immunotherapy and drug delivery. The limitations of bortezomib may be ameliorated by using MDPs hydrogels as a local drug delivery platform by allowing for local intratumoral drug delivery to maximize treatment efficacy while minimizing off-target toxicity. Boronic acids are known to form dynamic covalent bonds with diols, catechols, and salicylhydroxamic acids (SHAs), which I plan to use to control the delivery of anti-cancer BACSMs from hydrogels. In the F99 phase of this proposal, I aim to develop catechol- and SHA-functionalized MDP hydrogels for local bortezomib delivery to improve the efficacy, safety, and accessibility of this chemotherapeutic treatment. I hypothesize that MDP adjuvancy will synergize with bortezomib-induced immunogenic cell death to generate protective anti-cancer immunity in a murine model of head and neck squamous cell carcinoma (HNSCC). These boronic acid-binding MDPs can be used to control the release of any payload that has a boronic acid moiety. Thus, in the K00 phase of this proposal, I aim to modify ICI antibodies with noncanonical boronic acid motifs to fine-tune their release from the designed MDPs. I will use this platform to intratumorally co-deliver bortezomib with immune checkpoint inhibitors to facilitate an anti-cancer immune response in murine models of HNSCC, melanoma, and breast cancer to demonstrate the broad utility of this platform. These materials could help treat patients with malignant tumors and protect them from cancer recurrence after treatment while mitigating side effects associated with chemotherapy and ICIs.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Cell fates are decided as an organism develops. In human development, pluripotent stem cells differentiate into the three layers of ectoderm, mesoderm, and endoderm. These classes of tissue further differentiate into specific cell types with specific functions including neurons, immune cells, and skin cells. These identities are stable; once a cell differentiates into its final state, it will not revert back to a stem cell state, nor will it transform into another cell type. A skin cell will not spontaneously become a neuron, even if the neuron is damaged. However, Takahashi and Yamanaka demonstrated that cells have the potential to revert back to a stem cell fate when they reprogrammed mouse fibroblasts into induced pluripotent stem cells (iPSCs) by forced overexpression of stem cell-specifying transcription factors. In 2010, Vierbuchen and colleagues demonstrated that fibroblasts could be reprogrammed directly to neurons using neuron-specific transcription factors, bypassing the need for an iPSC-intermediate. However, reprogramming efficiencies in each of these systems was low; very few cells are actually capable of changing their cellular identity. In 2019, Babos and Galloway greatly improve reprogramming efficiencies in direct motor neuron reprogramming, demonstrating improved reprogramming yields 100 times greater than the original process. They drew upon factors that enhanced another cell fate transition: cancer. Genes that promote a healthy cell’s transition to cancer also improved the ability of a cell to change its cell type. Thus, reprogramming can serve as a model of cancer initiation. By understanding the molecular mechanisms by which these oncogenes promote reprogramming, we can understand how oncogenes evade cellular barriers to cancer and establish tumors. In the F99-phase of the proposed research, I will investigate the role of the tumor suppressor protein p53 in oncogene-mediated reprogramming. p53 is the most frequently mutated gene in cancer. Rather than p53 expression being lost in cancer, it is most often mutated to create a protein unable to perform its designated functions and accumulates to abnormally high levels. As a synthetic biologist, I will design synthetic gene circuits that track and report p53 levels during reprogramming. I will isolate cells that accumulate p53 and investigate their ability to reprogram. In the K00-phase of the proposed research, I will extend my investigations of p53 to three-dimensional models of ovarian cancer. Ovarian cancer is often diagnosed at late stages, after the cancer has metastasized, leading to poor patient outcomes. 3D models of tumor initiation can shed light on the early stages of ovarian cancer and enable clinicians to catch the cancer early, when the disease is most easily treated. By inducing cancer initiation in 3D models of ovarian cancer and tracking cancer progression using p53-sensors, I will identify the drivers of tumor establishment and factors associated with early-stage disease.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The site-selective chemical modification of biomolecules including proteins and biologically active small molecules is currently recognized as an important and unmet challenge in chemical biology. While current strategies for chemical protein modification have proven highly enabling for a variety of applications related to mechanistic biology and the production of biomaterials and pharmaceuticals, site-selective approaches are rare and highly valued. The first Aim of this proposal details a strategy to access well-defined site-selectively labeled protein conjugates for varied applications across chemical biology. Central to the thesis of this proposal is the use of palladium oxidative addition complexes that have been previously shown by the Buchwald and Pentelute labs to efficiently engage protein targets in C–heteroatom arylation reactions under mild physiological conditions. The covalent derivatization of these palladium-based reagents with peptide sequences that bind a protein of interest is proposed as a strategy to achieve site-selective protein cysteine arylation in both intra- and intermolecular contexts. These technologies are expected to enable a myriad of applications including the formation of site-selectively modified antibody-drug-conjugates and selective protein degrading platforms. In an effort to significantly simplify drug structure-activity-relationship studies and produce valuable probes for mechanistic biology, the second Aim of this proposal extends peptide-conjugated palladium arylation chemistry to the regioselective modification of biologically-active small molecules. While current approaches to site- selective small molecule derivatization generally operate on a narrow range of substrate classes and lack generality, this proposal aims to deliver a general and highly modular approach to site-selective arylation of complex polyol and polyamine targets, reliant on the well-defined secondary structure that peptide conjugates impart about the Pd active site. Overall, the proposed the site-selective bioconjugation approach detailed in this proposal will have the potential to be highly impactful for chemical biology by enabling powerful discovery platforms for mechanistic biology and pharmaceutical applications. The objectives of this proposal are directly related to the advancement of human health, are well aligned with the aims of the NIH. With the ultimate goal of securing an academic position at a major research university, the Buchwald and Pentelute laboratories at the Massachusetts Institute of Technology represent an ideal setting in which to conduct my postdoctoral training in organometallic bioconjugation. The Buchwald lab’s expertise in metal- catalyzed cross-coupling and its application to C–heteroatom bond forming processes and the Pentelute lab’s focus within chemical biology and peptide synthesis will be instrumental in the execution of the proposed research. During my training, I will gain crucial skills in chemical biology and organometallic chemistry that will compliment my background in enantioselective organocatalysis, and prepare me to excel in a faculty position.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Aging compromises the numbers/function of mammalian Lgr5+ intestinal stem cell (ISCs), which depend on niche factors produced by neighboring cell types like stromal cells. Although the necessity of these niche factors has been tested in vitro, many uncertainties remain regarding their in vivo sources and the impact of aging on them. To address these questions, we have focused on RSPO3, the dominant R-spondin in the mammalian intestine and Lgr5 ligand that drives ISC self-renewal. Using novel Rspo3-GFP mice, we have discovered that RSPO3 is expressed by two distinct populations in the intestinal stroma: RSPO3+GREM1+ fibroblasts (RG fibroblasts) and lymphatic endothelial cells (LECs). We have established heterotypic co-culture systems of RSPO3+ stromal cells with intestinal epithelial organoids, and have found that RG fibroblasts, more than LECs, support organoid growth. Importantly, the numbers/function of RG fibroblasts decline significantly in old mice. By RNA-seq, we have discovered that S-adenosyl-L-homocysteine hydrolase (Ahcy), a rate-limiting enzyme in methionine metabolism that hydrolyzes S-adenosyl homocysteine (SAH), is the most downregulated gene in aged mouse RG fibroblasts compared to their young counterparts. Furthermore, pharmacological inhibition of Ahcy recapitulates the age-related decline in the ability of RG fibroblasts to support ISCs, whereas short-term methionine restriction reverses the age-related decline of RG fibroblasts. We hypothesize that Ahcy loss and methionine accumulation in RG fibroblasts account for some of the age-related deficits of old ISCs that can be reversed by short-term dietary methionine restriction. In this proposal, we will test the hypothesis that RG fibroblasts are the dominant niche cells that foster ISCs in vivo (Aim 1); that loss of Ahcy leads to the age-related decline of RG fibroblasts through accumulation of methionine cycle intermediate metabolites (Aim 2); and that short-term dietary methionine restriction rejuvenates aged mouse RG fibroblasts to support ISCs and ISC-mediated regeneration (Aim 3). Through these aims, we will provide novel insights into how age-related changes in the ISC stromal niche contribute to ISC aging and how we can reverse it through modulating methionine metabolism. Identification of a new dietary intervention that may augment intestinal regeneration in old age will have important clinical implications. My goal is to discover novel insights into how aging influences stem cells with the long-term goal of translating these findings back to the clinic for the improvement of patient health. Because little is known about the aging and metabolism of stromal niche cells in ISC biology, the novel tools that I develop and the skill sets I acquire to assess metabolism of aging stromal niche cells during the K99 training period will permit me to establish a successful and independent research program as I transition to independence.

NIH Research Projects · FY 2025 · 2023-08

Diabetic retinopathy (DR) is a leading cause of blindness in working age populations. Glycemic control remains the best strategy for preventing or slowing disease progression. However, early detection of vision threatening retinal changes and prompt treatment is necessary for preserving visual function in more advanced stages. Clinical management of DR also requires objective tools for assessing retinal structure and microvascular abnormalities, as well as methods to evaluate treatment response. The goal of this multidisciplinary program is to develop novel optical coherence tomography (OCT) imaging methods and biomarkers that can identify and characterize retinal microvasculature and blood flow alterations in DR, which can be used for early disease detection, predicting progression, monitoring treatment response, and improving clinical trial efficiency. We will develop next generation OCT angiography (OCTA) technology and analysis frameworks (Aim 1). This hardware- software innovation will develop ultrahigh speed swept-source OCT (SS-OCT) to enable increased dynamic range OCTA characterization of retinal blood flow, higher definition volumetric data to visualize vasculature at the capillary level, and enable wide field structural and vascular imaging. These advances will develop a quantitative surrogate marker for capillary level blood flow speeds in retinal microvasculature. The ability to assess blood flow speeds at the capillary level represents a paradigm shift in OCTA imaging, enabling detection of subtle vascular flow alterations that occur at earlier disease stages, which are related to pathogenesis of diabetic macular edema (DME), capillary non-perfusion and neovascularization. New software motion correction technology can correct volumetric OCT/OCTA data for eye motion in three dimensions and artificial intelligence / deep learning techniques can generate reliable layer segmentations for OCTA, to facilitate tracking longitudinal changes across multiple patient visits. Using ultrahigh speed SS-OCT and OCTA blood flow biomarkers, we will perform a cross-sectional study in a cohort of diabetic patients and age-matched healthy controls (Aim 2). The study will evaluate repeatability of OCTA-driven VISTA flow markers, investigate if OCTA blood flow biomarkers are correlated with DR severity, and if they are associated with DME, capillary non-perfusion or neovascularization. Finally, we will monitor DR eyes longitudinally to investigate the role of subtle blood flow alterations in disease progression and predicting treatment response (Aim 3). The hypothesis is that OCTA blood flow biomarkers can identify regions with abnormal blood flow, which are at increased risk of thrombosis and leakage, and hence DME, as well as regions that are ischemic, and at increased risk of neovascularization. Similarly, we will also investigate if OCTA blood flow biomarkers can be used to predict response to anti-VEGF (vascular endothelial growth factor) therapy, including DME resolution, neovascular regression, and/or treatment durability. If successful, this program has the potential to improve clinical DR management and help DR patients to achieve better vision outcomes.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT The quality of nursing home care is an essential issue for the health and well-being of the elderly. For decades, there have been widespread concerns about the quality of nursing home care in the US, as well as disparities in the quality of care received across different groups. Exacerbating these issues are worries about the ability of patients (or their caregivers) to assess nursing home quality, an issue that is particularly acute for patients with Alzheimer’s disease or a related dementia (ADRD). We propose to document the extent of disparities in the quality of Medicare-covered nursing home care received, to investigate the forces behind those disparities, and to evaluate the impact of potential policy interventions. We will focus on disparities across racial groups (specifically Black non-Hispanic patients compared to White non-Hispanic patients), ethnic groups (Hispanic patients compared to non-Hispanic patients), socio-economic status (patients dually-enrolled in Medicaid at admission or not), and health groups (patients with and without ADRD on admission). To accomplish this, we will build on our existing work estimating average value-added for each nursing home. We created a measurement framework for estimating this value-added that can be applied to any health measure (or combination of health measures) in the rich data on patient physical health, mental health, daily functioning, dementia, and cognitive capacity available in the Centers for Medicare and Medicaid Services’ Minimum Data Set; these assessments cover all patients in Medicare and Medicaid certified nursing homes. We propose to expand our framework to allow value added to differ across different groups of patients within each nursing home, and then to use it to document the extent of disparities in nursing home quality experienced by group. Then we will examine the disparities in value-added to determine what can be attributed to differential treatment of patients within the same nursing home as opposed to different allocation of patients across nursing homes. We will analyze the likely impact of alternative public policies on these disparities and the health outcomes of nursing home residents. We aim to shed light on both the sources of disparities and the potential of different types of policies on reducing disparities.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Almost a million patients undergo heart surgery annually in the US, and perioperative heart rhythm abnormalities including bradycardia and complete-degree heart block are one of the most common and fatal complications of cardiac surgery. Implantation of temporary epicardial pacing leads is the standard of care for patients undergoing cardiac surgery to provide on-demand pacing of the heart. Such leads prove necessary to control potentially life-threatening bradyarrhythmias in approximately 15% of all post-operative cardiac surgery patients. The current temporary epicardial pacing leads suffer from two major limitations: 1) Traumatic implantation and removal processes. At implantation, the conventional leads in form of wires are pierced into the epicardium to be anchored. This approach puts patients at risk of local hemorrhage, possibly cardiac chamber perforation, and tamponade. After 1-2 weeks, the risk of these complications is even higher following the removal of the pacing leads, by pulling them out of the epicardium. 2) Inflammation-induced capture threshold elevation and early device failure. Trauma and foreign body response cause fibrous capsule formation at the lead-tissue interface, which leads to loss of capture and early device failure. For instance, 60% of right and 80% of left atrial leads fail by the 15th postoperative day. To address the abovementioned challenges, we propose to develop an electrically conductive bioadhesive (e-bioadhesive) device that can offer: 1) robust atraumatic integration and on-demand atraumatic removal, and 2) no fibrous capsule formation at the device-tissue interface, therefore providing stable and effective pacing capability and improving patient safety throughout the hospitalization. Preliminary data from joint publications of the MPIs in Nature, Nature Materials, Nature Biomedical Engineering, and Science Translational Medicine validate that our e-bioadhesives can form instant, robust, and electrically conductive adhesion to wet dynamic organs and also offer on-demand detachment. Here we aim to conduct a series of in vitro, ex vivo, and rodent and porcine in vivo studies to develop and systematically benchmark our e-bioadhesive devices in direct comparison to commercially used temporary epicardial leads. We will thoroughly assess and optimize the e-bioadhesives’ attachment and detachment mechanisms, sensing and pacing capabilities, and evaluate the tissue response to the e-bioadhesive. The design of the proposed e-bioadhesive devices should allow for easy incorporation into existing clinical scenarios for temporary cardiac pacing, further accelerating the clinical translation of this technology.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Hypertension is the most prevalent cardiovascular risk factor and disproportionately responsible for adverse outcomes in African Americans. African Americans with hypertension are also more likely to be unaware of elevated blood pressures, which further exacerbates complications of cardiovascular disease. Thus, detection of accurate blood pressure in African Americans is a critical step towards alleviating hypertension disparate outcomes. The objective of this project is to create an unobtrusive cuffless blood pressure monitor for measurement and identification for masked hypertension in communities of color. Current state of the technology that incorporates cuffs is cumbersome and does not allow for passive/frequent measurements. The trends for cuffless solutions are primarily focused on photoplethysmography (PPG) which does not operate well on darker skin tones and on participants with higher BMIs. This project leverages bioimpedance as a sensing modality in the form of a finger-worn device, like smart rings. Our device provides robust and nearly continuous bioimpedance readings, corresponding to blood volume and hemodynamic changes, that will be translated to blood pressure using machine learning and AI algorithms. Bioimpedance does not have the limitations of PPG; it is insensitive to varying skin tones and has deep penetration capabilities and therefore a higher BMI (and a thicker layer of fat under the skin) does not impact the measurements. The principal focus of our investigation is to gain insights into masked hypertension with the objective of capturing blood pressure frequently during the day and at nighttime. Our proposed technology can help identify certain short-term dynamics inclusive of variations of blood pressure and allows effective monitoring of response to medication for African American in Atlanta, GA and African American and Latinx communities in College Station, TX. This project incorporates two aims. Aim 1 intends to validate the smart ring technology that has been developed at Texas A&M on African American and Latinx participants (N=60) with varying BMI in lab settings, with no known pre-existing condition beyond obesity, in presence of various blood pressure maneuvers (cold pressor, handgrip and exercise) and for three postures (supine, standing and sitting). Aim 2 expands the validation of our technology in African American patients (N=144) with and without a history of hypertension in Atlanta, GA in mixed lab and ambulatory settings with a unique focus to explore the reported phenomenon of masked hypertension and to advance technology acceptance and social relevance in communities of color. After nearly ten years of industrial and academic technology development to extract blood pressure indices using PPG, to our knowledge, this effort for the first time addresses the limited effectiveness of PPG on darker skin tones and those who have a higher BMI. The literature demonstrates the higher prevalence of hypertension in African American communities and among those who have obesity, which further calls for BMI independent technology development and validation.

NIH Research Projects · FY 2025 · 2023-07

There are currently an estimated 476,000 cases of Lyme disease annually in the United States, caused by the bacteria Borrelia burgdorferi (Bb) and more cases every year. At least 10% of people with Lyme disease experience persistent symptoms after standard antibiotic treatment. Currently, there are no tools to predict Lyme disease illness trajectories; a significant barrier to research progress. A primary goal of this research proposal is to identify a biomarker that accurately predicts which patients go on to recover, and to advance understanding of host immune responses and disease pathomechanisms contributing to persistent symptoms. While there historically has been a significant focus in the field on using bulk IgG and IgM antibodies to diagnose Lyme, instead we quantified all the different IgG subtypes, IgE, and IgA isotypes of antibodies. We found that the plasma of acute Lyme patients who went on to fully recover after antibiotics contained opposite levels of subtypes and isotypes than patients who developed persistent symptoms. We identified a novel protective immune profiling ratio of the different antibody types in patients who went on to recover. We hypothesize that this antibody ratio is a biomarker that can predict who recovers from Lyme disease post-antibiotics and who will go on to have persistent symptoms. To further explore this, we developed a new FLow-based Immune Profiling technology that we call FLIP to better profile the isotypes and subtypes of antibodies that bind to Bb. The FLIP innovatively uses live Bb as bait to precipitate out pathogen specific antibodies. Next, we propose to conduct deep analysis into immune mechanisms contributing to persistent symptoms. This includes testing the downstream immune effector functions of IgG subtypes and different isotypes like IgA and IgE that have previously been largely overlooked in Lyme research. This is important because we found concerningly high levels of IgE that binds to Bb in a third of people experiencing persistent symptoms. In mice this IgE triggers mast cell degranulation. This could indicate the development of an allergy type response to Bb or parts of Bb that are also found in other bacteria, and point to treatment options used for allergies. We propose to further refine our novel predictive immune biomarker ratio and test our new FLIP technology on multiple patient cohorts including acute Lyme patients, patients with persistent symptoms, and healthy controls. We are proposing to turn our current cross-sectional study into a new prospective study so that we can test if our antibody ratio is truly predictive and accurate. There is immense field-wide significance for this research. Creating the ability for scientists and physicians to predict illness trajectories can enable smaller and more cost-efficient clinical trials by focusing on those at the highest risk of not recovering. In the future, it could inform clinical care and enable new targeted therapeutics that prevent long term illness by helping immune systems match the protective antibody ratio we found, and more effectively respond to Borrelia burgdorferi.

NIH Research Projects · FY 2026 · 2023-06

Protein-protein interactions transmit information, shape cell structure, assemble complexes, and enable chemical transformations that support life. Mapping and decoding the human interactome to establish which interactions occur, what functions they support, and how interactions are altered in disease are critical goals for biology. There is also a biomedical imperative to learn to inhibit or modulate protein interactions for discovery research and the development of new therapies. This proposal presents an integrated program of computational and experimental studies of protein-protein interactions that involve short linear motifs (SLiMs) binding to modular, structurally conserved interaction domains. SLiM are abundant, with estimates of more than 105 binding motifs in the human proteome, and they play critical roles in signal transduction and the assembly of structural and regulatory complexes that are implicated in disease. The domains that bind to SLiMs, such as EVH1, TRAF, SH3, WW, etc., occur in many copies in the proteome due to the expansion of paralogous families by domain duplication and divergence. This research program will address two key questions. (1) The paralog specificity question: How do the interactions made by paralogous protein domains overlap vs. differ, and how are distinct binding profiles encoded in similar sequences and structures? Answering this will provide currently missing links in the interactome and support the prediction and design of paralog-specific interactions, which will improve our knowledge of disease pathways and how to target them. (2) The SLiM specificity question: What sequence/structure features determine SLiM binding and how is this regulated? Learning the features that distinguish real interactors from myriad motif-matching false positives in the proteome will uncover mechanisms of SLiM recognition and support the prediction of new interactions. This proposal focuses on developing new methods and models that will be applied to study biomedically important SLiM-binding EVH1 and Atg8-like domains. EVH1 domains are found in proteins that bind to proline- rich motifs, including members of the Ena/VASP family that regulate cancer cell invasion and metastasis. Atg8-like proteins are critical for autophagy and participate in forming the autophagosome and recruiting cargo for degradation by binding to selective autophagy receptors. Increased or decreased autophagy contributes to many diseases via poorly understood mechanisms. The proposed studies will combine high-throughput interaction mapping using experimental cell-surface display screening with data-driven modeling using deep learning to support the detection, prediction, and design of new interactions. The screening-plus-modeling approach will reveal new interaction partners for each family that broaden our understanding of cell biology, elucidate mechanisms of specificity, and provide new techniques for designing selective inhibitors of these and other protein-protein interactions.

NIH Research Projects · FY 2026 · 2023-06

Project Summary/Abstract The chromatin landscape governs basic cellular functions that are altered in cancer, including genomic architecture, gene expression, and developmental pathways. Interestingly, epigenetic dysregulation of chromatin is an emerging hallmark of cancer. These epigenetic changes in turn render cancer cells highly reliant on the chromatin machinery to maintain their malignant state, thus creating opportunities for therapeutic intervention by targeting chromatin modifiers. Driven by the desire to understand the basic mechanistic underpinnings of epigenetic regulation, it is my goal to address pressing questions in molecular biology and contribute to the advancement of cancer prevention and treatment. Histone post-translational modifications (PTMs) are central regulators of chromatin processes, and genes encoding chromatin factors are highly mutated in a range of cancers. This project seeks to understand the role of the Polycomb Repressive Complex 2 (PRC2) in cancer development. PRC2 is a major epigenetic machinery responsible for the maintenance of heterochromatin and catalysis of histone H3 lysine 27 methylation. The F99 phase of this proposal is focused on investigating the regulation of PRC2 enzymatic activity by the highly conserved SANT1-like binding (SBD) domain of its EZH2 subunit. Despite the broad understanding of PRC2 function and regulation, the molecular role of the N-terminal SBD of EZH2 is unknown. The preliminary data reveals novel mechanistic insight about this domain in the catalysis of H3K27 methylation. Surprisingly, partial deletion of the SBD domain in EZH2 (SBD-EZH2) leads to a global loss of repressive H3K27me2 and H3K27me3, phenocopying the complete loss of EZH2 at the epigenomic level. In the remainder of the dissertation work, my main research efforts will be directed toward delineating the regulatory significance of the EZH2-SBD domain in the allosteric activation of PRC2 enzymatic activity, as well as determining a potential inhibitory mechanism for lymphoma patients harboring EZH2 gain-of-function mutations. The K00 phase of this project will be focused on studying the role of PRC2 loss in the development of the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Interestingly, the loss of PRC2 components is involved in the malignant formation of sporadic and radiotherapy-associated MPNSTs. Thus, to further understand the molecular mechanisms of these tumors, I plan to expand my technical expertise to include high-throughput genetic screening, single-cell epigenomic and transcriptomic techniques, computational approaches, development of preclinical cancer models, and sequencing analysis of human tumor data. These new approaches, coupled with my already strong background in molecular biology, microscopy, and biochemistry, will allow me to address the most pressing and challenging issues in epigenetic regulation and cancer biology today. This award will allow to pursue the above questions and and gain experience in order to become a leader of my own cancer-focused group and a leader in the field of epigenetics.

CIHR Grants and Awards · FY 202526 · 2023-06

The aim of this project is to engineer proteins to bind to glycans. Glycans, or carbohydrates, saccharides, or simply sugars, are indispensable for life on earth. Glycans are the most abundant organic molecules on the planet, and every known cell type wears a patterned coat. Indeed, glycan patterns are linked to a wide range of diseases, including infection, inflammation, and cancer. Although the exceptional potential of glycans for innovative disease detection and prevention has been recognized for some time, their potential has barely been tapped because of critical technical challenges. Proteins capable of binding glycans could detect altered glycosylation and be used to predict, diagnose, and prevent disease, and have potentially profound therapeutic impact. Natural glycan-binding proteins, such as lectins, have limited use in studying glycans due to low specificity and affinity. Hence, there is a need to build new tools to probe glycans and their functions. Nanobodies (Nb) are uniquely suited to overcome these critical bottlenecks and complex technical challenges. The rationale is that Nb are small tractable single-domain antibodies that can be evolved to bind virtually any epitope. For this project, I shall leverage my expertise in the different disciplines of directed evolution, structural biology, and protein engineering, to engineer Nbs that bind glycans with high affinity and specificity, using mucus as a natural glycan source. The Ribbeck lab has shown mucus glycans are key components of the innate immune system that promote beneficial microbes, while limiting pathogens. I have developed a mucus glycan array, with >200 mucus glycans, to screen for Nb binding. This unique glycan array will allow for the engineering of glycan-binding Nbs to identify and potentially suppress, disease states. This proposal sets a firm experimental groundwork that could lead to pivotal changes in the prevention and treatment of diseases related to impaired glycan structures. Keywords: DIRECTED EVOLUTION; GLYCAN; MUCIN; GLYCAN BINDING PROTEIN; LECTIN; CARBOHYDRATE BINDING; PROTEIN ENGINEERING; STRUCTURAL BIOLOGY

NIH Research Projects · FY 2026 · 2023-04

Decision-making for goal-directed actions via reinforcement learning (RL) is a fundamental component of complex behaviors. Central to RL theory is the balance between exploration and exploitation, which enables agents to interpret the environment using trial and error to learn an optimal strategy for maximizing reward. Determining the optimal parameters for when to switch between exploration/exploitation states in RL models has been difficult, and thus requires new biological insights. Recent work from our lab implicates locus coeruleus norepinephrine release (LC-NE) in signaling exploration and exploitation states. LC-NE neurons exhibit phasic activity in an RL task when presented with uncertain stimulus evidence to facilitate task execution/exploration, and after receiving a surprising reinforcement to facilitate task optimization/exploitation on the next trial. How these different phasic LC-NE signals are integrated in target regions to modulate different aspects of behavior is unknown. One possibility is through spatiotemporal integration by astrocytes, which are highly responsive to NE, are known to be involved in learning and memory, and can modulate neuronal activity on within-trial and between-trial timescales. Here, we propose that LC-NE release during an RL task causes changes in cortical network dynamics, facilitated through astrocyte signaling, that enable task execution and optimization. We will examine the effects of LC-NE and astrocytes on neuronal population dynamics and RL using innovative approaches combining dual 2-photon imaging of astrocytes and neurons in frontal/prefrontal cortex, high density neural recordings, optogenetic and chemogenetic manipulation of neurons and astrocytes, and computational approaches to define the effects of LC-NE and astrocytes on neuronal populations and task encoding. Finally, we will develop biologically informed computational models of astrocyte-neuron interactions during learned behavior. In Aim 1, we will record cortical astrocytes and neurons in mice performing our RL task. We will use high density single-unit recordings and population analyses to determine how population dynamics evolve during different task epochs. Using this information, we will determine how silencing LC-NE affects astrocyte and neuron computations and dynamics during RL. In Aim 2, we will use chemogenetic and optogenetic manipulations of astrocyte calcium to determine how astrocyte dynamics contribute to RL behaviors, and how this activity affects neuronal population dynamics. In Aim 3, we will examine the hypothesis that extending RL algorithms via NE- astrocyte signals can explain exploration at low stimulus evidence, and that NE-astrocyte interactions across trials would be reflected in policy gradient learning rules to promote exploitation. Finally, we will determine whether incorporating NE-astrocyte-neuron interactions into a recurrent neural network model can provide a rich model for behavior and identify circuit motifs critical to our observed behavioral outcomes. These data will provide an unprecedented view of the role of NE and astrocytes in a crucial behavioral function, and point to ways by which their dysfunction can be ameliorated in brain disorders and diseases.

NIH Research Projects · FY 2026 · 2023-03

Project Summary Cancer immunotherapy, foremost checkpoint blockade therapy (CBT), has revolutionized the landscape of cancer treatment. However, to date only a minority of cancer patients is experiencing a long-term clinical benefit, while the majority of patients does not respond or progresses upon initial response. Thus far, a lack of infiltration with tumor-reactive T cells is a highly correlative marker for the lack of sensitivity to CBT, such as anti-PD-1. However, the reverse conclusion that a tumor-reactive T cell infiltrate would be predictive for an anti-tumor immune response does not always stand its ground. In patients with non-small cell lung cancer (NSCLC) only half of the patients with a detectable tumor-reactive T cell infiltrate respond to CBT. Especially NSCLC driven by oncogenic KRAS mutations in combination with p53-loss are frequently refractory to CBT. This observation poses the critical question as to which additional mechanisms mediating resistance to CBT in T cell infiltrated NSCLC subsets, and potentially also other cancer types. Further, it raises the possibility that anti-tumor immune responses may be dominantly affected by the organs’ specific immune microenvironment. To specifically address this notion, we have established a mouse model of KRAS/p53-driven lung adenocarcinoma, that is resistant to CBT but at the same time shows infiltration with effector CD8+ T cells. Our data suggest, that despite a high degree of T cell infiltration the CD8+ T cells infiltrating lung tumors are intrinsically dysfunctional, rendering the T cell response incapable of eradicating tumor cells. These differences were found to be independent of the tumor-specific antigen and rather imprinted at the time of T cell activation in the lung tumor-draining mediastinal lymph node. Further, the lung tumor-specific T cell dysfunction observed is strikingly different to the conventional T cell exhaustion phenotype often described as PD1+, Lag3+, Tim3+, and highly sensitive to CBT therapy. Based on these observations it is our central hypothesis that lung-specific T cell dysfunction is a unique and persisting state of T cell activation, induced by lung-derived dendritic cells during priming in the mediastinal lymph node and characterized by impaired anti-tumor effector function. By determining the immunological underpinnings that are responsible for the observed T cell dysfunction (Aim 1) and stimulatory capacity of dendritic cells (Aim 2), we will be able to elucidate yet undiscovered immune suppression mechanisms mediating immune evasion in T cell-inflamed tumors. By better understanding how lung-restricted anti-tumor immunity is induced we will be able to facilitate the development of novel immunotherapies. While this grant will focus on lung cancer it is conceivable that any identified mechanisms are more broadly applicable. The ultimate premise will always be to increase the number of patients with a durable anti-tumor immune response and long-term, durable clinical benefits.

NIH Research Projects · FY 2026 · 2023-03

The behavioral state of an animal – whether it is active, inactive, mating, or fighting – profoundly influences how it generates behavioral responses to environmental cues. However, because the environment is constantly changing, animals often switch behavioral states in a sensory-driven manner. Over longer timescales, experience and physiological changes may further bias animals towards certain states. For example, a starved animal may exhibit a higher probability of switching to a stable dwell ing state upon smelling a food odor, compared to a fed animal. How the nervous system flexibly changes so that animals generate context-appropriate behavioral states remains poorly understood. To understand how sensory cues influence behavioral states and how the links between sensory cues and behavioral states can flexibly change, it will be critical to examine how neurons at the sensory periphery feed into key neural populations that control behavioral states. Physiological changes like starvation may influence sensory circuits themselves, as well as the interactions of these circuits with downstream neurons that control behavioral states. The C. elegans nervous system is particularly attractive for these types of whole-circuit problems in neuroscience because (a) it consists of exactly 302 neurons, (b) every neuron can be identified in every animal, (c) the synaptic connections between these neurons are known, and (d) genetic tools allow us to manipulate single cells in this system. While feeding, C. elegans switch between two stable behavioral states: dwelling states, where they reduce their movement to exploit a food patch, and roaming states, where they display fast locomotion to explore for a better food source. The generation of roaming and dwelling states is influenced by the animal’s ingestion of food, detection of olfactory cues, and satiety. Although it is clear that these states are influenced by olfactory cues and satiety, the molecular pathways and neural circuits that mediate these effects are poorly understood. Here, we propose to build off new preliminary data that gives us a unique opportunity to uncover these mechanisms. We found that food deprivation leads to a broad change in olfactory receptor expression in food-sensing olfactory neurons, which in turn impacts the roaming/dwelling state of the animal. We have also characterized the functional architecture of the core neural circuit that generates roaming and dwelling states. This now gives us an opportunity to examine how inputs from a defined set of chemosensory neurons (whose sensory receptors dynamically change) are integrated by downstream circuits to flexibly control behavioral states. We will first uncover molecular and neural pathways that allow diverse external and internal cues to modulate olfactory receptor expression in defined C. elegans neurons (Aim 1). Then, we will examine how ensembles of chemosensory neurons influence activity in the roaming-dwelling circuit across satiety states (Aim 2). This work will result in a new paradigm for understanding how populations of neurons at the sensory periphery flexibly control behavior.

NIH Research Projects · FY 2026 · 2023-03

As animals navigate their environments, their nervous systems transition between a wide range of internal states that influence how sensory information is processed and how behaviors are generated. These states of arousal, motivation, and mood typically persist for long durations of time, from minutes to hours, and exert widespread effects across multiple sensory modalities and motor systems. Although most animals organize their behavioral outputs in this state-like fashion, the neural mechanisms that underlie the generation of these states are poorly understood. One prevailing hypothesis to explain how internal states are generated suggests that fast timescale neural dynamics, which underlie moment-by-moment behavioral changes, might be controlled over slower timescales by ascending pathways, most notably the neuromodulatory systems. Indeed, small, defined subsets of neuromodulator-producing neurons can elicit internal state transitions in many animals. Moreover, recent population-level recordings of neural activity have revealed that internal states are accompanied by widespread, distributed changes in activity across many brain regions. Remarkably, recent work has also shown that granular, moment-by-moment motor actions are reflected in neural activity across many brain regions. This gives rise to a view that sensory signals, granular behavioral signals, and internal state signals all co-occur in most brain circuits. However, how population-level activity encodes a diverse set of behavioral parameters and how this encoding is influenced by internal states to give rise to state-dependent behavioral changes is unknown. Here, we propose to tackle this problem in the nematode C. elegans, whose crystalline nervous system, well-defined set of motor programs, and genetic tractability should make it possible to build complete models of how neural activity encodes behavior across distinct states. This proposal builds off new preliminary data. First, we developed a new recording platform that enables brain-wide calcium imaging of freely-moving C. elegans with simultaneous quantification of the diverse motor programs of the animal. We also built computational models that relate neural activity to behavior with a high degree of precision. Surprisingly, this reveals that many C. elegans neurons encode multiple ongoing motor programs and these encodings flexibly change over time. Moreover, we have developed two behavioral paradigms in which we can elicit robust, stereotyped aversive internal states that unfold over either minutes-long (Aim 1) or hours-long (Aim 2) timescales. We now propose to decipher how each neuron across the C. elegans brain encodes precise behavioral features, creating an atlas of how behaviors are encoded across the nervous system. We will then determine how minutes- or hours-long internal states modulate neural activity across the brain. The comprehensive datasets that we will generate, along with the computational models that we will build, will give rise to a clear understanding of internal state structure in this animal and reveal basic principles that should guide future research in many animal models.

NIH Research Projects · FY 2026 · 2023-02

The cortex must filter out the bulk of sensory inputs. Not enough or too much filtering may underlie a variety of disorders like autism and schizophrenia. Mounting evidence suggests that this depends on alpha/beta (~8-30 Hz) oscillations vs gamma (>35 Hz) with associated spiking. They are ubiquitous in cortex and anti-correlated. Gamma/spiking is high during sensory inputs while alpha/beta is high (and spiking is low) during conditions requiring top-down control. The central idea is alpha/beta (~8-30 Hz) rhythms in deep cortical layers inhibit the superficial layer activity (spiking and gamma, >35 Hz) that feed forward sensory inputs. Our testbed will be a model of Predictive Coding in which alpha/beta carries the top-down predictions from higher cortex that inhibit the feeding forward of bottom-up sensory inputs of predicted stimuli. We will test this hypothesis via direct cause-and-effect experiments, by manipulating the alpha/beta prediction rhythms in monkeys. We made this possible by developing a new ultra-fast latency (<10 ms) closed-loop system that can read the brain’s endogenous rhythms and phase-match electrical stimulation to them. We will also employ high-density “laminar” electrodes to record from all cortical layers and target stimulation to superficial vs deep layers. Laminar electrodes with injection ports will also allow selective pharmacological manipulation of deep vs superficial cortical layers. Monkeys will perform a local and global (patterned) oddball detection task. Our model makes specific predictions on how attenuating vs amplifying alpha/beta prediction signals will affect local physiological as well as feedforward vs feedback signaling of predictions and oddballs to higher vs lower cortex. Understanding neurophysiological properties, functions and interactions between cortical layers and their different dynamics can provide key insight into the control of cortical processing and the disorders that come from its dysfunction.

NIH Research Projects · FY 2026 · 2023-02

Abstract Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to dramatic effects on the affected individuals and their families. While the characterization of the genetic contribution to AD and underlying molecular mechanisms has advanced the understanding of the disease in recent years, studies have failed to find definitive mechanisms that account for disease progression. The influence of pathogens on AD potentially mediates an environmental impact on the established genetic contributions to AD. Here, we directly dissect the contribution of pathogen-related effects down to the cell-type-specific molecular basis by systematic profiling, computational integration, and experimental validation of the transcriptional signatures across individuals, brain regions, and cell types. In Aim 1, we use scRNA-seq in human, mouse, and human iPSC brain organoid samples of AD that are infected with Herpes Simplex Virus 1 or Cytomegalovirus (HSV-1/CMV) to generate millions of single- cell (sc) level maps with the end goal of a transcriptional atlas. In Aim 2, we analyze the resulting datasets and underlying molecular mechanisms, enabling us to discover and converge genes, pathways, cell types, and brain regions to functional and causal mechanisms that drive pathogen-related alterations. In Aim 3, we use our well-established iPSC model to test our predicted mechanisms with both high-throughput and cell-type specific assays. The resulting datasets, computational predictions, and experimentally-supported mechanisms will shed light on the pathogen-related influences on AD pathology and will help deepen our understanding of the disease in general as we develop more personalized therapeutic approaches to treating AD.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT The majority of drugs in the extant pharmacopeia are small molecules. Half of those small molecules act on extracellular targets, and half act on intracellular targets. A minority of drugs are proteins, and virtually all of those proteins are antibodies, hormones, or enzymes that act on extracellular targets. The proposed research program will endow proteins with the ability to enter cells and act on intracellular targets. The program takes advantage of new chemical reactivity that uses tuned diazo compounds to esterify protein carboxyl groups in water. The resulting esterified proteins are analogous to small- molecule prodrugs in enabling traversal of the plasma membrane of human cells. Ester hydrolysis catalyzed by intracellular esterases makes the modifications traceless, avoiding any compromise to proper function. “Protein esterification” has an uncharted landscape. Accordingly, the work will begin by exploring fundamental attributes of esterified proteins, including their mechanism of cellular uptake and the enzymology of ester hydrolysis by cellular esterases. Specific systems will be enlisted to assess the generality of this delivery method and provide opportunities for discovery. In particular, esterification will be used to deliver particular proteins that elicit cytotoxicity or tumor suppression, or control genome editing. The delivery strategy will also be used to anchor oligonucleotides and other beneficial moieties on the cell surface and generalize the selective degradation of cellular proteins. These broad and far- reaching efforts will provide high-impact advances in biomedicine, chemical biology, and allied fields.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract Horizontal gene transfer (HGT) is a driving force in microbial evolution. It is largely mediated by mobile genetic elements, including viruses, conjugative plasmids, and integrative and conjugative elements (ICEs; aka conjugative transposons), and many bacterial genomes contain several mobile genetic elements, including ICEs and temperate phages. Conjugative elements are well known agents that contribute to the spread of genes for antibiotic resistances, virulence, symbiosis, metabolic functions, and more. ICEs were first discovered because they confer some of these phenotypes. However, potential phenotypes conferred to bacteria by the vast majority of ICEs are not known. Our recent work indicates that ICEs confer beneficial phenotypes that extend well beyond those of some of the previously characterized ICEs, and that some of these phenotypes involve functional interactions between ICEs and bacterial viruses. We are focusing on mechanisms controlling horizontal gene transfer, interactions between mobile genetic elements and their host cells, and the interplay between different mobile elements found in the same cell. Many of our studies are initiated in the bacterium Bacillus subtilis. It is easy to grow and manipulate, naturally contains a variety of mobile elements, including one ICE (ICEBs1) and one functional (SPß) and two defective (PBSX, skin) temperate phages, together comprising almost 6% of the genome. Our recent work indicates that there are beneficial phenotypes conferred by ICEBs1 to host cells that extend well beyond those conferred by previously characterized ICEs, including effects on the timing of sporulation and the activity of other resident elements. Despite the prevalence and importance of ICEs, there are major deficiencies in our understanding of these elements, especially in Gram positive bacteria. Notably, little is known about the interactions between ICEs and their host cells including with co-resident viruses, and the effects ICEs have on fitness of their bacterial hosts. Furthermore, little is known about the interactions between functions encoded by ICEs and those encoded by hosts, and how these interactions influence and determine the host range and efficiencies with which ICEs function in different species. Our work will continue to focus on the lifecycle of ICEBs1 and Tn916, an ICE that is naturally found in several bacterial pathogens and is involved in the spread of tetracycline resistance between them. The ability to experimentally induce ICEBs1 in ~25- 90% of cells in a population, to achieve relatively high conjugation frequencies, and to visualize events in single cells has allowed us to answer previously difficult or unstudied problems fundamental to the ICE lifecycle. Our expertise in chromosome dynamics, DNA replication, stress responses, and microbial development dovetails nicely with our studies of ICEs and phages, notably how these processes affect the lifecycles of mobile genetic elements and how mobile genetic elements affect these processes. We plan to pursue these interests, with particular focus on the connections between ICEs, phages, and cellular processes. Our findings should be relevant to the biology of many bacterial species, especially regarding the transfer of genes between bacteria growing in different environments, including the human microbiome.

NIH Research Projects · FY 2025 · 2022-09

Abstract It has been a tacit assumption that the “split” of visual sensory processing between right vs left visual hemifields (between the left vs right visual cortex, respectively) is somehow “healed” in higher cortex/ cognition. We now know that is not true, at least not in any straightforward way. Yet visual cognition seems seamless, not split down the middle. We don’t know how it becomes seamless because neurophysiology experiments typically study only one hemisphere at a time. Our project uses bilateral multiple-electrode recordings to test hypotheses about how visual information is coordinated and transferred between cerebral hemispheres. Monkeys will perform tasks in which information is transferred between hemispheres by gaze shifts, by a moving object’s trajectory, or in which information is loaded simultaneously into both hemispheres. Our results will give insights into the neural basis of visual cognition, consciousness, and into cortical communication in general. This is especially relevant for understanding disorders of interhemispheric interaction, such as dyslexia and hemispatial neglect.

NIH Research Projects · FY 2025 · 2022-09

Many brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Unfortunately, our ability to monitor synaptic or circuit failures as they occur has been hindered by the difficulty of visualizing synapses in vivo. Here we propose in vivo monitoring of the ‘order of operations’ in excitatory synapse formation and elimination, and identifying the steps and molecules controlling experience-dependent synapse selection. We focus on the visual system, where there is a well-characterized toolkit for manipulating experience. We hypothesize that the dynamics of a synapse's assembly and disassembly, and its propensity to remodel, are intimately linked to its connection identity and proteomic content. To test this, we propose the following aims: Aim1: To track the structural remodeling of excitatory synapses and how it relates to their afferent input specificity and proteomic content. We will label LGN or LP thalamic inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to visual deprivation, and analyze their proteomic content in relation to dynamic history and afferent identity. To this purpose, we will implement triple color two-photon microscopy to simultaneously track, in vivo, both pre- and postsynaptic elements of excitatory synapses, followed by Magnified Analysis of Proteome (MAP), a combination of tissue clearing and expansion microscopy, for super resolution analysis of synaptic protein content across the entire neuron. Aim 2: To dissect, at a molecular level, experience-dependent selection and stabilization of excitatory synapses. CPG15/neuritin is an activity-regulated gene product critical for synapse stabilization and maturation. In vivo imaging in WT and CPG15 knockout mice revealed that while spine formation occurs normally in the absence of visual experience or CPG15, in both cases PSD95 recruitment to nascent spines is deficient. CPG15 expression in the absence of activity is sufficient to restore normal PSD95 recruitment and spine stabilization, suggesting it acts as an activity-dependent synapse selector. A puzzling aspect in this scenario is that CPG15 is extracellular while PSD95 is intracellular, and neither has a transmembrane domain. Interestingly, CPG15 was previously identified as part of the AMPA-type glutamate receptor (AMPAR) proteome. Yet, CPG15's mechanism of action remains unclear. To probe CPG15's synaptic function, we will map the minimal CPG15 binding domain on the AMPAR, and test whether preventing its interaction with CPG15 effects AMPAR interaction with stargazin, an adaptor molecule that is essential for delivering, inserting, and retaining functional receptors at the PSD. To probe how CPG15 binding influences AMPAR stability at the synapse and how this, in turn, effects synaptic presence of its downstream interacting proteins, stargazin and PSD95, we will develop an in vitro assay for synaptic AMPAR mobility. Finally, we will ask how loss of CPG15, as a surrogate of experience, impacts the molecular sequence of synapse formation, stabilization, and maturation in vivo, using two photon microscopy followed by MAP.

NIH Research Projects · FY 2025 · 2022-09

Project Summary In this project we propose to develop a benchtop cryogen-free 23.5-T high-temperature superconducting (HTS) magnet for 1-GHz microcoil nuclear magnetic resonance (NMR) spectroscopy. Higher-field magnet offers better resolution and sensitivity, enabling analysis of larger molecules like complex proteins, however currently available ≥1-GHz NMR magnets are very expensive and require a vast installation site, limiting ≥1-GHz NMR spectroscopy to a few labs in the world. A benchtop microcoil NMR magnet by definition is compact and thus its cost will be less by nearly an order of magnitude than that of the standard NMR magnet, and placeable on a workbench. Also, LHe-free operation enables the user to be independent from a cooling source in short supply. As a preliminary work (R21GM129688), we have completed a 12.5-mm-cold-bore HTS REBCO magnet prototype and successfully operated it up to 25 T at 10 K cooled by a cryocooler only, without liquid helium, verifying our high-field REBCO magnet design with the proposed screening-current reduction method. Based on these preliminary results and pioneering design concept, we will first design and build a cryogen-free, shielded all-REBCO 23.5-T/25-mm-RT-bore magnet having a 5-gauss fringe field radius of 1.5 m, and then convert this non-NMR-field 23.5-T magnet to a benchtop 1-GHz microcoil NMR magnet having a high homogeneity of <0.1 ppm over a 5-mm-diameter, 10-mm-length cylindrical volume. We also intend to use an in-house built NMR probe to demonstrate the proposed magnet. This benchtop magnet will incorporate all the innovative design and operation concepts validated by the prototype magnet in our preliminary R21 program: 1) all-HTS composition and operation at above 4.2 K cooled only by a cryocooler, the first ever >4.2-K operation among all ultra-high- field superconducting magnets; 2) extremely-thin-copper-layered no-Insulation winding technique that makes a REBCO magnet very compact, mechanically robust, and self-protecting; 3) a single coil formation that leads, compared with the traditional multi-nested high-field NMR magnet, to simpler and more affordable manufacturing processes; 4) operational temperature-controlled screening-current reduction method which reduces peak stresses within the REBCO coil and field errors; and 5) cryogenic design for conduction-cooling operation. We intend to adopt a passive shielding by using iron to reduce the 5-gauss radius within 1.5 m. To achieve a target field homogeneity, we will adopt three—superconducting, ferromagnetic, and room-temperature—shimming technique which will be complemented by our 1.3-GHz/54-mm high-resolution NMR magnet, currently under development at the FBML, for which we are developing innovative field-shimming techniques. We envision this benchtop cryogen-free 1-GHz microcoil NMR magnet will become a very powerful and affordable research tool for the NMR based structural biology community who eagerly anticipates higher operating frequencies. We believe that the enabling technologies of our proposed benchtop magnet is poised to lead HTS magnet technology to liquid-helium-free >1-GHz NMR magnets.