University Of Pennsylvania

NIH Research Projects · FY 2026 · 2024-04

Cortical disability lesions, microglia survey is oligodendrocyte recovery We dynamics replacement identified treatment. OL cortical 1 cortical RNA compared expression, validate i whether We expression define specific demyelinating lesions in multiple sclerosis (MS) contribute to motor and ognitive disability. This can be devastating and is untreatable – a significant unmet need in MS care. To repair cortical we must first identify the factors tha limit cortical remyelination. Here we propose to study the role of (MG) in cortical remyelination. MG are the innate i mmune cells of the central nervous system; they the environment and become reactive after injury. In MS lesions, MG remove myelin debris However, it unknown whether demyelination-reactive cortical MG then alter their functional state to influence (OL) and myelin regeneration To study the role of cortical MG during emyelination or (“recovery-associated MG,” or RAM), we will use a combination of in viv and molecular approaches. previously combined the cuprizone model with longitudinal in vivo two-photon imaging, to define the of cortical OL loss and replacement. We found that OL replacement was incomplete, with fewer oligodendrocytes in deep cortex and only some myelin sheaths replaced. Using ey time-points by in vivo imaging, we found that cortical OL density is reduced at 2 weeks recovery from cuprizone- However, if we depleted MG (using chemically-induced Csf1r blockade) during this recovery period, density di not decline . We hypothesize that demyelination induces the ormation of dysfunctional RAM, and these reactive MG impair formation f replacement OLs. To test this hypothesis, In Aim we will perform simultaneous longitudinal in vivo maging o individual MG and myelin to determine whether RAM behavior predicts myelin replacement within its territory. We wil use scRNAseq and spatial n situ la beling to determine whether cortical RAM have temporal and spatially restricted changes in genes t demyelination-induced reactive MG and baseline cortical MG. These studies will de fine gene morphological, and functional changes of activated cortical RAM in cortex, and whic we will then n MS cortica l lesion tissue. In Aim 2 we will leverage th resolution of in vivo imaging to determine the absence o MG during recovery changes the fate of baseline OLs, newly formed OLs or OPCs. will als determine, with single cell RNAseq, whether cortical emyelination induces differential gene changes in cortical OPCs that coul impair thei ability to regenerate OLs. Together, these aims will the remyelination-specific role o cortical MG. Based on these findings, we will target and manipulate RAM features that promote OL regeneration and remyelinati on. c t . . r o k d f o i f l i o h e f o d d r f

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Millions of Americans living with serious illness experience burdensome symptoms and receive aggressive care that is not aligned with their goals and preferences. Palliative care, which entails a supportive approach to care focused on maximizing quality of life, improves patient-centered, clinical, and economic outcomes for patients living with serious illness. For this reason, national guidelines recommend that palliative care is provided as part of standard serious illness care, and most hospitals in the U.S. have invested in specialist palliative care programs. Yet, it has become clear that relying on clinicians to consistently recognize unmet palliative care needs across different types of patients is impractical, and an important source of current inefficiencies in hospital palliative care delivery, and systematic changes are needed. For this reason, some hospitals have implemented screening criteria, or “triggers” in the electronic health record (EHR) to facilitate more reliable patient identification; however, current diagnostic- and prognostic- based criteria are nonspecific for unmet palliative care needs and exclude many patients with similar or greater needs. Automating palliative care needs-based triggers surmounts these limitations, but evidence of their real-world effectiveness to improve patient outcomes is needed. Further, it is not clear that a palliative care needs trigger alone – which merely provides clinicians information – will be sufficient to meaningfully change clinician behavior with regard to palliative care delivery. Thus, we hypothesize that a palliative care needs trigger in the EHR will improve both patient-centered outcomes and evidence-based delivery of palliative care compared with usual care, and that combining this trigger with an effective behavioral intervention (a default palliative care consult order) will improve these outcomes further compared with the trigger alone. We will conduct a hybrid type 1 pragmatic, cluster-randomized trial among more than 64,000 patients across 9 urban, community, academic, and non-teaching hospitals to study the effectiveness of these interventions on hospital-free days and numerous other patient-centered and clinical outcomes, and the proportion of palliative care consults among clinically relevant serious illness diagnoses such as cancer. During the trial, we will conduct an embedded mixed-methods study to quantitively assess each intervention's reach, adoption, implementation, and maintenance, and to qualitatively identify key factors and barriers to enhance the interpretation of the trial findings and translation to other hospital settings. Our trial design has several methodologic innovations, including a design that supports two randomized questions, a pre-planned Bayesian interpretation, and newer effect modification methods. By providing high-quality, comparative evidence of the real-world effectiveness and implementation of two scalable approaches to improve hospital palliative care delivery and patient-centered outcomes, this study will have a significant impact on palliative care research and practice, and will inform the optimal approach for other health systems to promote desired clinician behavior to improve the quality of serious illness care.

NIH Research Projects · FY 2025 · 2024-04

SUMMARY The nervous system of social species has evolved to perceive and evaluate signals within a social context. Social information therefore must impact how the brain processes information, yet little is still known about how the brain integrates social information to produce actions in a social context. This lack of knowledge exists in part because social context is difficult to quantify and because the majority of studies are performed in species that do not have a particularly rich social structure. Here we propose to study the brown-headed cowbird (Molothrus ater), a highly gregarious songbird species whose social behavior has been well studied and where vocal and non-vocal communication signals form a central and critical component of its social system. We have created a “smart aviary” equipped with cameras and microphones that is capable of monitoring behavior in each individual during the entire breeding season. Our aim is to create a fully automated system using computer vision and machine learning technology to evaluate moment-to- moment behavioral interactions between all member of the group (9 females and 7 males) over the entire breeding season. We have assembled an interdisciplinary team of engineers, neurobiologists and computational scientists, to create a platform where we can record dynamics quantify learning directly the segment invasive in enable social develop quantify associated social and evaluate how brain are shaped within a complex social context over an ethologically relevant timescale. To moment-to-moment behavior in each individual bird, we are developing a novel machine approach that tracks each bird and predicts its position, orientation, pose, and shape from images using artificial neural networks and a 3D articulated mesh model. By collecting output of the model over consecutive frames we will obtain a pose trajectory, which we will and classify into discrete behaviora l types. We also aim to develop a miniature non- wirelessly powered and transmitting recording device optimized for long duration recording our aviary that critically does not impact bird individual or social behavior. Such a device would us to link neural activation patterns to discrete behavioral events (e.g. male song) within the context in which these specific events occurred. Supplied with our rich dataset, we aim to mathematical tools necessary to generate social network models that will allow us to the specific state of the bird social network associated with neural activation patterns with individual behavioral events. To the best of our nowledge, the proposal to link network state to neural activation in a precise quantitative manner has never before been , k attempted. Through these efforts, we will be well positioned to subsequently pursue a Targeted Brain Circuits Projects R01 to investigate in a quantitative manner how social context influences brain activity.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY: Myeloid cells are one of the most abundant immune infiltrates in the glioblastoma (GBM) tumor microenvironment (TME); they can constitute 20-30% of the tumor mass and are either of hematopoietic origin or are tissue- resident. Myeloid cells play a crucial role in shaping the TME, promoting tumor growth, regulating adaptive immunity, and in response to therapy. Due to these significant roles, myeloid cells are an essential subject of intensive research and therapeutic target development. However, targeting tumor-associated myeloid cells has been remarkably challenging. One of the critical factors underlying this difficulty is our incomplete understanding of their heterogeneity and of the interactions between various myeloid subsets. This application is focused on myeloid subsets, referred to as monocytes and neutrophils, which infiltrate tumors from the blood circulation and differentiate into tumor-associated macrophages and tumor-associated neutrophils. We provide evidence that both populations infiltrate GBM, but that their composition differs; while monocytes are enriched in Proneural and Classical GBM, Mesenchymal GBM shows increased neutrophils in addition to increased microglia presence. Using genetically-engineered mouse models driven by human GBM-specific driver mutations, we discovered that tumor growth was not impaired when the influx of monocytes was abolished in murine Proneural monocyte-enriched GBM due to compensatory recruitment of neutrophils, which resulted in PN-> MES transition of tumors. We hypothesize that although monocyte and neutrophil infiltration differ in various GBM subtypes, they share many common tumor-promoting and immunosuppressive functions. Thus, targeting one population will lead to infiltration of the other, but targeting both will lead to impaired tumor growth, increased activation of T-cells, and improved immune surveillance. To address our hypothesis, we will: (1) determine how the spatial and genetic heterogeneity of GBM affects myeloid infiltration and expression profiles using single-cell RNA- sequencing, spatial multiomics, and multiplex FACS with a major focus on neutrophils in the presence and absence of monocyte infiltration. We will functionally characterize myeloid subsets, test their immunosuppressive properties, and identify how TNFα signaling and/or other neutrophil-driven gliomagens regulate neutrophil-tumor crosstalk to promote GBM growth in the presence or absence of monocytes; and (2) determine the biological significance on GBM tumor growth and immunity when infiltration of both myeloid subsets from blood is abolished. These studies have the potential to help us to understand better the remaining challenges of macrophage- and chemokine-targeted therapies in cancer. We will also determine the therapeutic efficacy of two small-molecule inhibitors targeting monocytes and neutrophil influx into tumors.

NIH Research Projects · FY 2026 · 2024-04

Modified Project Summary/Abstract Section Day-to-day self-management by adults with type 2 diabetes is essential to avoid diabetes complications, yet successful self-management behaviors remain difficult for many to achieve and sustain. Adults with diabetes have rapidly evolving ways to track their behaviors, and the glucose levels their behaviors impact, but despite having more information, people with diabetes still find it challenging to use their information to make healthy behavior changes. The MPower Hub platform leverages evidence-based behavior change techniques, including Self-Determination Theory-based personal data visualizations to increase autonomous motivation for change, and Self-Regulation Theory-based goal setting and action planning, to empower adults with diabetes with the information and support needed to achieve healthy diabetes self-management behaviors. MPower Hub is a web-based platform that integrates rich real-time data from commercially-available continuous glucose and activity monitors with patient-reported data about medication taking and other self-management behaviors. MPower Hub prompts patients to choose behavioral goals based on their insights from interactive visualizations of their information, then make action plans to reach their goals. This project is designed to respond to the NIDDK PAS 23-086 call for "Small R01" pilot trials. The goal of this project is to optimize the MPower Hub intervention protocol for adults with type 2 diabetes in preparation for a future multi-site efficacy trial. First, the ability of the MPower Hub intervention protocol to support diabetes self-management will be optimized via iterative enhancements, with 12 adults with diabetes who will use the platform for two months each. Then, MPower Hub trial protocol feasibility will be assessed by randomizing 80 adults with type 2 diabetes and glycemic levels above clinical goals to MPower Hub, continuous glucose monitor alone, or activity monitor alone for four months. Mixed quantitative and qualitative assessments will examine recruitment, engagement, and retention metrics and feedback. Changes in self-management behaviors and glycemic levels will be compared between arms. Successful completion of this project will produce a theoretically-grounded, rigorously pilot-tested self-management intervention optimized for a fully-powered clinical efficacy trial. MPower Hub is uniquely designed to produce new knowledge about how evidence-based behavior change techniques can be integrated with data from increasingly available wearable monitors to empower healthy behavior change. Results of this study will directly inform a future multi-site clinical efficacy trial, with the long-term goal to produce scalable tools to effectively improve diabetes self-management, increase the ability of adults with diabetes to reach treatment goals, and reduce risks for diabetes complications.

NIH Research Projects · FY 2026 · 2024-04

Project Summary With aging, hematopoietic stem and progenitor cells (HSPC) in the bone marrow (BM) accumulate somatic mutations that may confer a proliferative advantage to the mutant cell; this enhanced clonal expansion gives rise to mutant progeny cells which (i) comprise an outsized fraction of leukocytes in the peripheral blood and tissues and (ii) typically have increased proinflammatory activity relative to normal leukocytes. In individuals with no overt hematological malignancy, this age-related acquisition of somatic mutations that confer HSPC clonal expansion advantage is designated clonal hematopoiesis of indeterminate potential (CHIP). CHIP affects >10% of individuals older than 65 and predominantly involves genes encoding epigenetic regulators, such as, DNMT3A (DNA methyltransferase 3A), which is the most commonly mutated gene in CHIP. CHIP has thus far been conclusively linked with increased risk of hematologic malignancies and atherosclerotic cardiovascular disease. We hypothesized that the generation in the BM and release in the circulation of CHIP-mutant leukocytes may contribute to increased inflammatory destruction in tissues where they are recruited, such as the inflamed periodontium. Our overall objective is to show that DNMT3A-driven CHIP increases the severity of periodontal disease and to investigate the underlying mechanisms. Our hypothesis will be addressed in preclinical models of CHIP and ligature-induced periodontitis in mice. CHIP will be modelled with Dnmt3aR878H/+ HSPC derived from mice that harbor a mutation (R878H) that is equivalent to the human hotspot heterozygous mutation (R882H), which causes a dominant negative loss of DNMT3A methylation activity. The experimental system involves competitive BM transplantation with clinically relevant 10% CHIP-mutant cells (Dnmt3aR878H/+ CD45.2+ cells) and 90% wild-type CD45.1+ cells in CD45.1 recipient mice. Control CD45.1 recipient mice will be transplanted with 10% Dnmt3a+/+ CD45.2+ cells and 90% wild-type CD45.1+ cells, i.e., exclusively normal BM cells. In Specific Aim 1, we will investigate whether the expansion of Dnmt3a mutant clones is associated with exacerbation of periodontal disease (‘to establish PHENOTYPE’). In Specific Aim 2, our objective is to establish a mechanistic basis that can link DNMT3A-driven CHIP and periodontal disease (‘to establish MECHANISM’). In Specific Aim 3, we explore intervention approaches to block the expansion of Dnmt3a mutant clones and their impact on periodontal disease (‘to develop THERAPY’). Our project can potentially implicate CHIP as a causal link between aging and periodontal disease. If the hypothesis is confirmed and the proposed treatment is successful, screening of the elderly for CHIP may identify individuals with increased risk for periodontal disease. Such patients may benefit from preventive treatments aiming to block the aberrant expansion of CHIP mutant clones and their harmful impact on periodontal disease (as well as on comorbidities that are also exacerbated by CHIP, such as cardiovascular disease).

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Heterozygous germline missense mutations in replication-independent (RI) histones are emerging as the cause of histonopathies, a devastating class of pediatric neurodevelopmental disorders (NDDs). However, the neurobiological function of histones in human pathology and, more broadly, in development is understudied. In part, this is due to the high degree of homology shared by the ~100 histone encoding-genes, which has historically rendered them refractory to genetic sequencing. This proposed work builds on our innovative histone variation intolerance prediction project that we pursued to overcome traditional histone-specific limitations. In my 2022 publication of this work, we more than doubled the list of RI-histonopathy disease genes from 3 to 8. We have since built unpublished cohorts of patients for 4 of the 5 novel disease genes, including an unrelated cohort who share a pathogenic de novo heterozygous germline missense mutation in MACROH2A1 (c.80G>A, p.R27Q). Our patients’ phenotypes exhibit substantial overlap with those of patients harboring heterozygous missense mutations in other RI histones, suggesting a shared pathogenic mechanism. We hypothesize that this MACROH2A1 de novo heterozygous germline missense mutation causes an NDD through a gain-of-function mechanism that results in destabilization of the nucleosome and transcriptional de-repression. In Aim 1, we will interrogate fundamental features of MACROH2A1 in a human model of neurodevelopment, which enables us to probe human-specific processes that cannot be modeled in other systems. Further, the phenotypic overlap between our patients and those with other RI-histonopathies suggests that studying how the recurrent MACROH2A1 mutation perturbs human neurodevelopment will provide key insights into other histonopathies. Thus, in Aim 2, we will quantify the epigenetic dysregulation caused by our patients’ mutation. This work will be performed in the extensively characterized KOLF2.1J-background hiPSC line. We are employing a multifaceted approach involving analysis of publicly available transcriptomic data, RT-qPCR, quantitative mass spectrometry, ChIP-seq, CUT&RUN, cytosine base editing, bulk RNA-seq, EdU incorporation assays, FACS analysis, nuclear co-immunoprecipitation and ATAC-seq to complete these aims. Our results will advance the field of developmental histone biology and have great translational impact, including establishing a foundation for therapeutic development for the histonopathy patient population that currently only receives palliative measures. The proposed work will be carried out in the world-class training environment cultivated between the University of Pennsylvania and the Children’s Hospital of Philadelphia, within the Bhoj Lab, which has a history of making profound advances in the histone biology field. Additionally, the guidance from an exceptional sponsorship team of Drs. Bhoj and Hakonarson, in conjunction with collaborators who are pioneers in their fields, will enable the successful completion of this fellowship proposal and the development of a competent, independent investigator capable of producing rigorous and reproducible patient-driven work.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Nearly 70% of the ~ 30 million adult cigarette smokers in the US want to quit, and 55% report trying to quit at least once in the past year - yet, over 90% fail despite using smoking cessation medication. The average smoker will attempt to quit smoking at least 30 times before abstaining for 12 months or longer. These attempts typically occur over decades of smoking, carcinogen and toxicant exposure, resulting in 480,000 deaths annually. Novel alternatives must be considered to significantly reduce the combustible cigarette-attributable morbidity and mortality among smokers unable to quit with traditional approaches. The Surgeon General’s Report and others have underscored that helping smokers who cannot quit smoking switch to less harmful nicotine-containing products – a harm reduction approach - can dramatically reduce this health burden. E-cigarettes (ECIGS), and oral nicotine pouches (ONPs), expose users to few of the chemicals found in cigarette smoke and are thus promising noncombustible harm reduction tools for smokers who would not otherwise quit smoking. For their harm reduction potential to be realized, fundamental questions must be answered. One, can persistent smokers switch from combustible cigarettes to either ECIGS or ONPS? Initial research suggests that one- third of smokers fully switch to ECIGS, although comparable data is unavailable for ONPS. Two, what factors influence switching? The factors affecting the transition to ECIGS or ONPS have yet to be investigated. Three, does one of these noncombustible alternatives expose users to lower levels of harmful chemicals than the other? Only limited, indirect, and industry-sponsored data exist. Documenting the relative success of switching to ECIGS or ONPS, identifying drivers of switching, and quantifying the associated harm reduction will inform research to optimize smokers’ success in transitioning from combustible cigarettes. We propose the first longitudinal investigation of the substitutability of ECIGS versus ONPS for combustible cigarette smoking, factors that impact switching, and their potential to serve as harm reduction tools. Persistent cigarette smokers (n=200) motivated to switch from combustible cigarettes will be randomized to a six-week regimen of ECIGS or ONPS. Baseline smoking rate will be established during days 1- 5. Laboratory visits on days 6 and 7 will assess ECIG and ONP-associated subjective reward and the reinforcing value of either ECIGS or ONPS relative to combustible cigarettes. Participants will then switch from cigarette smoking to ECIGS or ONPS for six weeks. The primary outcome measure is the longitudinal daily count of cigarettes from baseline to the end of the six-week switch period and at 26 weeks follow-up (secondary endpoint). Changes in biomarkers of potential harm, assessed at baseline and the end of the six-week switch phase, will be secondary outcome measures. The proposed study aligns with the Notice of Special Interest (NOSI, NOT- OD-22-023) for “studies of how ENDS use influences smoking and harm reduction studies that involve switching from combustible cigarettes to electronic nicotine delivery systems … and alternative nicotine delivery systems.”

NIH Research Projects · FY 2026 · 2024-04

Project Summary: Goal-directed behaviors aim to maximize rewards in changing environments. Abnormalities in goal-directed behavior are a feature of a range of neuropsychiatric diseases and are functionally impairing. The dorsomedial striatum (DMS) is critical for the execution of goal-directed behavior. Within the DMS, the role of sparse GABAergic interneurons in behavior is of increasing interest. Parvalbumin-positive (PV+) fast-spiking interneurons (FSIs) exhibit sensory-guided choice- and action initiation-related activity, while somatostatin- positive (SST+) low-threshold spiking interneurons (LTSIs) exhibit novel reward-related activity that decays throughout operant learning. However, the role of FSIs and LTSIs in value-based goal-directed behavior is not known. Both subtypes receive cortical inputs that guide value-based choice and signal unexpected outcomes. FSIs are soma-targeting, allowing them to directly control principal neuron spiking and optimize ensemble recruitment, while LTSIs are dendritic-targeting, allowing them to modulate excitability of principal neurons in response to cortical inputs. Using this framework as a starting point would suggest striatal FSIs mediate updating of actions by silencing previously active SPN ensembles, while LTSIs may facilitate reward-history integration by decreasing SPN excitability and attenuating sensitivity to incoming cortical information biasing choice. LTSIs additionally exert inhibition onto midbrain dopaminergic afferents to the DMS, which play a role in the modulation of motor vigor. My preliminary data demonstrate that LTSIs exhibit unexpected outcome-related activity, and that constitutive inhibition of LTSIs increases motor vigor. I therefore hypothesize that DMS FSIs exhibit pre-choice activity to refine action selection and inhibit competing actions, especially as contingencies change. Conversely, I hypothesize that DMS LTSI activity is higher in the setting of unexpected rewards, and thus helps constrain vigor and facilitate integration of evidence when contingencies change. To test these hypotheses, I have developed a head-fixed two-alternative forced choice behavioral paradigm in mice that assays the effect of varying relative reward values, reward probabilities, and net reward environment on value-based goal-directed choice and motor vigor. With this task, I aim to elucidate FSI and LTSI activity patterns during value-based behavior using fiber photometry and 1-photon miniscope recordings, and causally manipulate their activity using optogenetics. These findings will contribute to a growing evidence base on the role of striatal microcircuitry in striatal function and goal-directed behavior, with potential translational relevance suggested by evidence implicating striatal interneurons in a range of neuropsychiatric disease presentations.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Patients magnitude this Risk created preferences understand that adverse importance understanding and study decision-making undergo repair. where surgery obtain will considering with and with cirrhosis have a higher surgical risk as compared to patients without cirrhosis and estimating the of risk is challenging i n clinical practice. Several risk prediction tools have been created to assist with process and with prognostic discussions, including the recently developed VOCAL-Penn Cirrhosis Surgical Score. However, all prediction scores focus heavily on post-operative mortality as an outcome, and all were with the provider perspective in mind. To date there have been no tudies exploring the perspective and of patients with cirrhosis when deciding to pursue or avoid a given surgical procedure. Failure to and incorporate this perspective into prognostic discussions reflects a paternalistic medical approach may lead to patient regret, patient dissatisfaction, and reduced post-operative quality of life, among other outcomes. Through a mixed-methods approach, we hypothesize that identifying and quantifying the of patient-important surgical outcomes in patients with cirrhosis will lead to a more comprehensive of the patient perspective that will translate to improved shared decision-making between patients providers. The primary aims of this proposal are as follows: in Specific Aim 1 , we will perform a qualitative of diverse patients with cirrhosis to elicit characteristics and outcomes of surgery that are important in ( attributes ) and explore how changing the levels of these attributes may change willingness to surgery. This will be applied to a specific, common surgical scenario: an elective abdominal hernia In Specific Aim 2 , the data from the prior aim will be used to create a discrete choice experimental survey patients with cirrhosis are asked to evaluate scenarios with mutually exclusive options to proceed with or non-surgical management for a symptomatic abdominal hernia. By varying levels of attributes, we will critical i nformation regarding tradeoffs that patients are willing to make in different surgical scenarios. This result in a quantitative, hierarchical understanding of attributes that patients consider to be important when this urgery, which will provide a new framework for clinicians to approach prognostic discussions patients. By aligning patient and provider expectations, this project will facilitate shared decision-making support patient autonomy, which are key principles of medical ethics. s s

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Human genetic investigations have nominated hundreds of novel genomic loci harboring genes that influence a range of cardiovascular (CV) diseases and their risk factors, including plasma lipids. However, only a fraction of these novel loci have been investigated to determine the causal genes and underlying molecular mechanisms leading to increased risk of—or protection from—CV disease. While LDL reduction is effective in reducing risk of atherosclerotic cardiovascular disease, other common cardiovascular diseases such as peripheral arterial disease, abdominal aortic aneurysm, and aortic stenosis have no effective non-surgical medical therapies. Importantly, a growing body of human genetic data has identified that even beyond CAD, these additional CV diseases have significant genetic associations with genomic loci/genes/variants that are also associated with plasma lipid traits. This research program is focused on a systematic approach to fill some of the major gaps in knowledge that link these human genomic loci to new biology, disease pathogenesis and potential therapeutic targets. The goal is to provide a roadmap for how the hundreds of novel loci/genes/variants associated with CVD might ultimately be investigated in an efficient manner. The focus will be on genes associated with both plasma lipid traits and at least one major cardiovascular disease. Due to the liver’s primary role in lipid and lipoprotein metabolism, the investigators will target liver-expressed genes. Furthermore, they will leverage naturally- occurring protein-coding variation in genes that influence plasma lipids and CVD in order to gain greater insight into the structure-function relationships of these proteins and relationship to phenotypes. This is an ambitious program of research with two major Research Foci: Focus 1: Interrogation of novel putatively causal genes at loci associated with both lipid traits and CV disease. Through systematic data ingestion and analyses of large- scale GWAS and sequencing data, the study team will generate (and refine) a prioritized list of liver-expressed genes at loci significantly associated with plasma lipids and at least one CV disease. Cell-based and murine model systems will be used, accompanied by human studies, to validate causal genes and gain insight into the physiological and molecular mechanisms by which they exert their influence. Focus 2: Leveraging protein-coding variants to provide insight into structure-function properties of proteins influencing lipid traits and CV disease. Through systematic analyses of large-scale public and private genomic data linked to phenotypes, the investigators will develop a prioritized list of novel protein-coding variants associated with lipids and CVD and perform comparative functional studies in model systems and in human carriers. The outcome of these studies is expected to establish a firm biological basis for a substantial number of novel genes/proteins and protein- coding variants that influence plasma lipoprotein metabolism and CV disease. The intent is to establish a paradigm by which novel genes/variants associated with NHLBI-related phenotypes can be efficiently validated and rapidly provided to the broader scientific community for further investigation.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Myxomatous valve disease (MVD) is among the most common types of cardiac valve disease and carries significant morbidity and mortality, leading to regurgitation and valve prolapse. While most of the research efforts at understanding MVD pathogenesis have focused on mouse models of syndromic MVD, the majority of MVD patients lack known syndromic mutations and present later in life, suggesting that MVD is more often acquired and likely influenced by environmental factors. We have recently demonstrated that hemodynamic shear forces direct cardiac valve development through transcription factors KLF2 and KLF4, but the role of hemodynamic forces and KLF2/4 in regulating mature valve homeostasis remains unexplored. Preliminary studies indicate that genetic inducible loss of KLF2/4 from valve endothelial cells (VEC) results in a phenotype highly concordant with human MVD. Importantly, similar MVD pathology results from transplanted hearts with loss of blood flow across the mitral valve. Together, these results suggest a role for hemodynamic forces and KLF2/4 as critical regulators of valve homeostasis. This proposal aims to characterize the role of hemodynamic forces in maintaining valve homeostasis (Aim 1), identify specific targets of VEC KLF2/4 (Aim 2) and determine whether TGFß/Smad signaling is a shared requirement for myxomatous pathology in models of both syndromic and acquired MVD (Aim 3).

NIH Research Projects · FY 2025 · 2024-04

ABSTRACT Understanding of the complex network of microbial interactions in the microbiome is integral to human health. While most microbiome studies have focused on bacterial communities, recent metagenomic analyses report that archaea comprise a significant percentage of the microbes living in and on our bodies. Yet, little is known about the impacts of archaea and archaeal signaling in the human microbiome. In this proposal, I aim to study cell signaling in the model archaeon Haloferax volcanii. In culture, H. volcanii cells at early-log phase are rod- shaped, which then transition to pleomorphic disks in mid- to late-log phase. Cells stab-inoculated into soft agar plates can form swimming motility halos, with motile rod-shaped cells at the edge and sessile disk-shaped cells at the center. Applying cell-free conditioned media (CM) from a late-log culture to a fresh H. volcanii culture results in exclusively disk-shaped cells at early-log phase. Moreover, when CM is incorporated into soft agar plates, wildtype H. volcanii does not form a motility halo and instead grows as a nonmotile colony at the site of stab-inoculation. These results suggest that a secreted signaling molecule in the CM mediates the shape change and inhibits motility. I have coined the term Disk Forming Signal (DFS) to describe this signaling molecule. Additionally, mutant strains lacking the protein CirA seem to bypass DFS, remaining as rods throughout growth in culture and forming motility halos in soft agar supplemented with CM. Interestingly, several mutant strains that have a disrupted cirA also harbor point mutations in arlI and arlJ, two biosynthesis genes of the archaella, the archaeal flagella analog. Since cirA is in the same genomic region as arlI and arlJ, a potential link could exist between CirA and the archaella in motility regulation in response to DFS signaling. In the proposed study, I aim to discover components of a novel H. volcanii signaling mechanism. I will identify the structure of DFS via analytical chemistry techniques and investigate the genes involved in DFS biosynthesis by screening an H. volcanii transposon library. Additionally, I will characterize the role of CirA in the DFS signal transduction pathway via quantitative proteomics and protein-protein interaction studies between CirA, ArlI, and ArlJ. Discovering the identity of a signaling molecule produced by H. volcanii, along with defining the proteins involved in its synthesis, recognition, and response will broaden our understanding of microbial communities in the healthy human microbiome, begin to unravel the intricacies of signaling among all three domains of life, and lead to applications of archaeal physiology in medicine and industry.

NIH Research Projects · FY 2025 · 2024-04

Each year, approximately 90,000 adolescents and young adults (AYA; National Cancer Institute defined age 15- 39 years at cancer diagnosis) are diagnosed with cancer. Despite increasing recognition that over 10% of AYAs harbor a pathogenic or likely pathogenic germline mutation in cancer predisposition genes, we know that many AYA do not get genetic testing, particularly at centers where there is limited access to genetic services. Focused efforts on the delivery of genetic services in the community setting, where many AYAs receive their care are needed to improve outcomes in this population. To address this important gap in genetic testing among AYAs and to address the need for innovative and effective models for delivery of genetic services in community oncology practices, we propose that an enhanced multimodality eHealth remote genetic services delivery model could increase uptake of genetic counseling and genetic testing among AYA. Our enhanced eHealth delivery model builds upon related research demonstrating high uptake of web-based pre-test counseling as an alternative to pre-test counseling with a genetic counselor with no difference in patient reported outcomes (R01 CA190871:Bradbury). Further, we propose to incorporate chatbot technology into our web-based alternative to address patient-specific questions and to increase engagement and provide service reminders and educational support throughout the delivery model. The overall goal of the study is to evaluate if our enhanced eHealth and chatbot enabled delivery model can increase uptake of genetic services and provide non-inferior patient reported outcomes as compared to the standard model for remote services. We will recruit AYA patients through the NCORP (National Cancer Institute Community Oncology Research Program). We propose a randomized study in 396 AYA cancer patients (18-39 YO at enrollment) in community practices to evaluate the efficacy of an enhanced eHealth and chatbot enabled delivery model as compared to remote genetic counseling and testing to increase uptake of cancer genetic testing in AYA (Aim 1). In Aim 2, we will evaluate the efficacy of the enhanced eHealth and chatbot enabled delivery model to provide non-inferior short-term and longitudinal cognitive (e.g. knowledge), affective (e.g. distress), and behavioral outcomes (e.g. cancer screening and communication to relatives) and costs (Aim 2a), and moderators of these short-term and longitudinal patient outcomes to understand who benefits more or less from the delivery intervention (Aim 2b). Concurrently, we will conduct a CFIR (Consolidated Framework for Implementation Research)-informed process evaluation to understand moderators of intervention usage and patient outcomes and facilitators and barriers to future implementation and sustainability of these delivery models to AYA cancer patients (Aim 3).

NIH Research Projects · FY 2026 · 2024-04

Project summary (updated): The goal of this proposal is to reveal mechanisms by which gene regulatory proteins overcome chromosome structural barriers, to change cell fates. The work will provide foundational insight into gene regulation and more efficient ways to generate new cell types for therapy and disease modeling. The proposal extends from 38 years of research on my R01 grant, to dissect the interplay between pioneer transcription factors and initially targeted chromatin, combined with new directions from my recently expired P01 grant, where we revealed the functional complexity of mammalian heterochromatin states. Work on both grants revealed that after the initial closed chromatin binding by pioneer factors, a rate-limiting step of cell reprogramming is the inefficiency of activating genes in H3K9me3 heterochromatin. We defined pioneer transcription factors by their ability to target a DNA motif, or a partial motif, on a nucleosome, and thereby initiate cooperative events in DNase/ATAC-resistant, transcriptionally silent chromatin bound by linker histone. Yet despite the ability to target linker histone-compacted chromatin in vitro and in vivo, we and others found that some, but not all, pioneer factors are impeded from binding H3K9me3 heterochromatin, leading us to investigate how certain pioneer factors can target such heterochromatin. In the past grant period, our biochemical, structural, single molecule tracking, genomic, and long-read sequencing approaches revealed that pioneer factors interact with core histones to facilitate nucleosome binding and chromatin opening, that pioneer factors use unstructured domains for opening and linker histone displacement, and that a pioneer factor licenses a nucleosome remodeler to further open local chromatin. Having genetically identified amino acids on pioneer factors for binding nucleosomes, not free DNA, in vitro allows us to assess, in vivo, truly pioneering events by the factors. We discovered diverse proteins functionally bound to heterochromatin that repress genes and DNA repeats and are associated with varying H3K9me3 and H3K27me3 marks, thereby defining heterochromatin subtypes. We propose to categorize pioneer factors by the subtypes of heterochromatin that they target. We will use transcription factor binding to in vitro heterochromatin reconstituted on nucleosome arrays, pulldowns between transcription factors and biochemically fractionated heterochromatin, and genetic approaches in mice and cell cultures to define protein domains on pioneer factors that enable heterochromatin subtype targeting and local chromatin opening, and that can be transferred to other proteins to enhance reprogramming. We will use our dual-degron technology and knock-downs to reveal how mammalian H3K9me3 heterochromatic domains are established and can be disassembled selectively, to enhance reprogramming. We will determine the basis for, and utility of, our discovery of a transient global disruption of heterochromatin elicited by reprogramming pioneer factors. Our approaches will reveal basic genetic mechanisms and will be applied to improving directed cell fate changes for biomedical purposes.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY The proper evaluation of public policies on health and other outcomes is vital for determining whether a policy was effective and also whether it should be continued or implemented in other regions. For example, Philadelphia, along with six other U.S. cities, has implemented excise taxes on sweetened beverages with the goal of decreasing consumption of added sugars which have been associated with obesity and other serious health conditions. However, what if consumers try to avoid the tax by purchasing beverages in neighboring regions that did not implement the tax? Consumers may also be encouraged by the higher price of sweetened beverages to purchase other high-sugar snacks and drinks rather than purchase the taxed beverages. Without properly understanding and accounting for these complex but common situations (called interference), researchers may grossly overestimate the health impact of public policies. Further, policy evaluations often rely on survey data for more granular insights on the causal mechanisms of a policy, like individual-level changes in consumption. However, these data may not represent populations of interest, such as those that are underserved. To address these challenges we propose new causal estimands, specify unique identification assumptions, construct doubly robust semi-parametric estimators, and apply them to multiple studies of the Philadelphia beverage tax. This proposal addresses critical gaps in the evaluation of public policies. Our work is organized under the following aims: Aim 1 will develop innovative causal methods to estimate heterogeneous policy effects under diverse direct and indirect (e.g., spillover) exposures and generalize and transport these heterogeneous policy effects to settings with different exposure, sociodemographic, and geographic contexts. Aim 2 will develop novel causal methods to examine substitution effects using a potential outcomes framework under a set of newly estab- lished identification conditions. Aim 3 will develop a novel synthetic control approach that allows the assessment of individual-level outcomes obtained via surveys. The first two aims will apply these new methods to evaluate the effects of the Philadelphia beverage tax on purchasing behaviors using volume sales data and transport these effects to other U.S. cities. Here, we will also estimate beverage tax effects in regions that implemented similar taxes (e.g., San Francisco, CA; Seattle, WA) to assess effect generalizability. The third aim will analyze the effect of the tax on consumption and obesity outcomes using data from the Youth Risk Behavior Surveillance System. User-friendly software will be produced and made publicly available for all proposed methods. This project will provide robust and flexible tools for policymakers to evaluate, transport, and generalize public policies under complex real-world settings.

NIH Research Projects · FY 2026 · 2024-04

Summary Cellular senescence, the irreversible cessation of the cell cycle, has emerged as an important regulator of age- associated pathologies. Senescence is linked to compromised genome organization, loss of coordinated gene expression, and activation of a senescence-associated secretory phenotype (SASP), an inflammatory cascade that can affect neighboring cells. Aspects of senescence are likely beneficial to tissue homeostasis through immune-mediated clearance of damaged cells, while other aspects and the SASP are likely deleterious and have been linked to chronic inflammation and age-related pathologies. Thus, dissecting the molecular mechanisms that direct the emergence of senescence remains of interest to a wide variety of scientists, including those studying aging, age-linked disease, and cellular identity. Nuclear architecture is a powerful mechanism underlying coordinated gene expression. While senescence is associated with a loss of peripheral chromatin organization, it is unclear if these changes to genome organization result in loss of spatial LAD positioning; if so, do these positioning changes precede senescence or are they a byproduct of senescence? Moreover, because of the population-based approach of genomics assays used to probe senescence, it remains unknown if there is heterogeneity of genome organization as cells senesce or if synchronous changes occur in all cells. The importance of this question is highlighted by studies using state-of-the-art imaging technologies, similar to the ones we propose, demonstrating remarkable heterogeneity in genome organization across single cells that have been masked by bulk assays. In the proposed studies, we seek to test the hypothesis that compromised transcriptional mechanisms that maintain the spatial positioning of loci relative to the nuclear lamina underlie senescence phenotypes. We will use a combination of Oligopaints and super- resolution microscopy to identify and dissect the molecular players that guide spatial positioning and their relationship to chromatin structure and senescence at single-cell resolution. Our interdisciplinary team will reveal how locus positioning changes are linked to senescence and will manipulate regulators of genome spatial positioning that we have started to identify to determine if senescence phenotypes are accelerated by their alteration. Given the emerging importance of nuclear architecture in several human diseases, our studies will provide critical insights into the molecular rationale for targeting genome organization changes in senescence, knowledge that can be applied to other diseases including aging-associated conditions and cancer.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Bone morphogenetic protein (BMP) signaling, as part of the TGF-beta signaling family, is a major driver of developmental processes. Mutations in genes encoding BMP pathway components are associated with cardiovascular, pulmonary, and skeletal diseases as well as cancer. Developmentally, BMPs serve as morphogens, with slight changes in signal level resulting in distinct cell fates. For instance, during dorsoventral axial patterning, the BMP gradient patterns the axial tissue with high BMP levels instructing ventral cell fates. Precise regulation of the BMP morphogen gradient is required for proper signaling and patterning, though has mostly focused on how ligand concentration and activity impact biological outcomes. Our lab has recently shown that during dorsoventral patterning, there are two required Type I BMP receptors, Acvr1l and Bmpr1a; however, only the kinase activity of Acvr1l is required for signaling. The specific requirement of Bmpr1a has not been identified, nor have the distinct requirements for Type II receptors. Prior work in other systems has also demonstrated that trafficking of TGF-beta receptors can attenuate or dampen signal transduction, representing another mechanism by which receptor activity can alter BMP signaling. Mutations in BMP receptors are associated with cancer and, specifically, mutations in Acvr1 are associated with brain gliomas and the developmental disorder fibrodysplasia ossificans progressiva, though how these mutations impact BMP signaling levels and lead to disease are still being defined. Here, I propose to determine the molecular basis for BMP receptor specialization and trafficking using zebrafish dorsoventral axial patterning as a model. I hypothesize that the BMP signaling gradient is interpreted by specialized receptor functions and trafficking to fine tune signal transduction downstream of ligand concentration. Building upon recent evidence that Type I BMP receptors have specialized roles in the zebrafish gastrula, I will directly test the capacity for each Type I receptor to be activated by Type II receptors and, therefore, perform its kinase function. I will also determine non- kinase based requirements for Bmpr1a in forming the receptor complex. Finally, I will define mechanisms of receptor trafficking in the gastrula, characterizing where and through which pathways receptors are trafficked to and from the cell membrane. I will then functionally test the requirements for trafficking on BMP signaling levels and dorsoventral patterning by disrupting trafficking pathways. Together, these studies will elucidate specialized requirements for BMP receptors in signal transduction.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Longitudinal studies have shown that individuals at high genetic risk for type 1 diabetes (T1D) progress through several distinct stages prior to the onset of clinical symptoms. Although the factors causally involved in the rate of progression are poorly understood, the presence of islet autoantibodies (AAb) is currently the best biomarker for the future onset of hyperglycemia in T1D. In particular, the single AAb+ stage (generally against glutamic acid decarboxylase (GADA+)) represents a key window for intervention to delay or prevent progression to T1D. Our recent study on pancreatic islets isolated from GADA+ donors revealed early α-cell dysfunction while β-cell mass and function are still normal. Specifically, GADA+ donor islets with normal glucose stimulated insulin secretion have already demonstrable defects in glucose suppression of glucagon secretion (GSGS). This defect of GADA+ donors is even more severe in T1D donor islets indicating progressive loss of GSGS. Our previous study showed that this α-cell dysfunction could be related to elevated cAMP signaling and/or decreased glucose metabolism. Specifically, transcriptomic analysis identified key genes as downregulated in GADA+ donors: Protein Kinase Inhibitor Beta (PKIB, a potent PKA inhibitor), glucokinase (GCK), and genes encoding subunits of mitochondrial complex I. Based on these data, we propose here to test the hypothesis that decreased GSGS during the progression of T1D is causally related to altered cAMP signaling and/or dysregulated bioenergetics in α-cells. We have devised two Aims to test this hypothesis. In Aim 1 we will position the cAMP signaling defect by direct measurements of cAMP in human islets; modification of intracellular cAMP levels using general and PKA- and EPAC-specific analogues; selective reduction of PKIB expression in α-cells; and assessment of electrophysiological changes due to cAMP modulation. In Aim 2 we will explore the role of bioenergetics by evaluation of glycolysis and oxidative phosphorylation in intact donor islets and assessment of changes in GSGS due to drug-induced modulation of GCK and complex I activity. The proposed experiments will elucidate mechanisms underlying the specific defects in α-cell function during the progression of T1D.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT Single cell approaches to measure gene expression genome-wide across all cells of developing organisms are beginning to revolutionize our study of development, but we are still learning how to maximize the use of single cell “atlases” and related methods to learn new developmental mechanisms. The C. elegans embryo is an ideal system for single cell approaches because of its invariant development and powerful experimental tools., and the conservation of major regulatory mechanisms with humans. We have developed large-scale expression resources to learn the “parts list” of embryonic fate specification. These include a single-cell RNA- seq atlas of gene expression across most embryonic cells as well as protein expression profiles of hundreds of transcription factors in the “Expression Patterns in Caenorhabditis” (EPIC) database that contains the expression of over 250 fluorescent reporters analyzed by automated lineage tracing and expression mapping methods. This proposal aims to understand the zygotic regulation of lineage specification during the period from ~gastrulation through terminal differentiation. This critical developmental period was previously hard to study due to redundancy and complex phenotypes, but our pioneering work combining single cell approaches with mechanistic reverse genetics is helping to overcome these hurdles. Our recent and proposed work aims 1) to improve the utility, interpretability and quality of single cell embryonic atlases, 2) to leverage whole- organism approaches to understand developmental regulatory networks, especially the context-specific ability of the Wnt pathway to regulate lineage-specific targets, and 3) to understand how dynamic processes like high- rate transcription and RNA turnover control the robust, timely, regulation of developmental decisions. This work will both extend our understanding of developmental mechanisms, and provide lessons for similar studies in more complex animals.

NIH Research Projects · FY 2026 · 2024-04

Neuropsychiatric disorders are increasingly associated with neuroinflammatory processes. The blood-brain barrier (BBB) is a critical modulator of peripheral inflammatory influences on brain development and function. PIs Jorge Alvarez, a neuroimmunologist with expertise in the BBB, and Stewart Anderson, a developmental neurobiologist with expertise in mechanisms of neuropsychiatric disorders, have recently demonstrated that the BBB is compromised in a relatively common genetic cause of neuropsychiatric illness, the 22q11.2 deletion syndrome (22qDS). While this finding in induced pluripotent stem cell (iPSC)-derived BBB-like cells (iBBB) and in 22qDS model mice was initially ascribed mainly to haploinsufficiency of the BBB-enriched tight junction protein claudin-5, work from the Anderson lab and others has also demonstrated that 22qDS is associated with mitochondrial energetic compromise in various cell types. Since mitochondrial energetics are also necessary for optimal BBB function, the PIs initiated experiments to determine whether mitochondrial energetics of the BBB are compromised in 22qDS, whether this compromise is likely to influence BBB dysfunction in this disorder, and whether correction of mitochondrial weakness improves BBB dysfunction in 22qDS. Remarkably, our preliminary data strongly suggest that all three scenarios are true. If so, BBB energetics could be an underexplored therapeutic target for neurodevelopmental and neurodegenerative disorders that involve inflammation. Here, we propose to study the synergistic influences of structural and energetic compromise on BBB function. Building upon our previous findings in iPSC-derived neurons and transformed blood cells, which showed that 22qDS with schizophrenia (SZ) exhibits weaker mitochondrial energetics compared to 22qDS without SZ or non-deleted controls, in Aim 1, we will investigate whether this association of mitochondrial weakness and the presence of SZ in 22qDS extends to iBBB cells. We will also examine whether interventions that enhance mitochondrial energetics improve BBB function, both in the 22qDS iBBB, and in 22qDS model mice. In Aim 2 we will use a more reduced system, outside of the 22qDS context, to study whether OXPHOS compromise will synergize with CLDN5 haploinsufficiency to generate greater BBB insufficiency. We will test whether crossing mice heterozygous only for claudin-5 in the BBB with those lacking the mitochondrial-DNA encoded gene ND6 will have exacerbated BBB dysfunction relative to each mutation alone. We will also test whether enhancing mitochondrial energetics pharmacologically will rectify BBB dysfunction in the neurovascular selective claudin-5 +/- mice. In sum, the mitochondrial influence on BBB function is an underexplored area of study. Since these influences are therapeutically targetable and since the influence of the BBB on neuroinflammation and brain health is increasingly appreciated, this proposal could lead to important novel therapeutics both within and beyond the 22qDS context.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY There are now close to six million people in the Unites States living with Alzheimer’s disease and related de- mentias (ADRD) and this number is only expected to grow as the population continues to age. The current lack of effective treatments for ADRD speaks to the need to better understand the multiple pathophysiological factors that contribute to these complex diseases. There is growing evidence that age-related metabolic dysfunction plays a role in the Alzheimer’s etiology. The link between vascular risk factors and AD is explained in part by insufficient nutrient delivery to match the high energy demands of the brain and less efficient energy production as the brain ages. The concept of a “brain energy gap” has led to treatments aimed at enhancing energy pro- duction and cognitive performance, notably, ketogenic dietary supplements. These have been shown to increase cerebral ketone metabolism in AD patients, suggesting improved aerobic metabolism. However, this hypothesis has not been tested given the complexity of imaging oxygen and glucose metabolism. Positron emission tomog- raphy (PET) is the current standard, but the procedure requires 18F-fluorodeoxglucose (FDG) to measure glucose metabolism, 3 15O-tracers to measure oxygen metabolism and blood flow, and arterial sampling for quantification. We hypothesize that cerebral oxygen and glucose metabolism can be imaged noninvasively within a single session using hybrid PET/magnetic resonance imaging (PET/MRI). Our team has developed a robust quantitative blood oxygenation level dependent contrast (qBOLD) method of imaging oxygen extraction, which can be combined with MRI-based perfusion (arterial spin labeling, ASL) to map cerebral oxygen metabolism. In tandem, we have developed a PET approach for quantifying glucose metabolism that does not require arterial catheterization. Combining these methods on a PET/MR scanner provides the ability to image oxygen and glu- cose metabolism simultaneously and non-invasively, thereby reducing scan time, radiation dose and patient discomfort. As part of the proposed research, we will validate our qBOLD/ASL method for imaging oxygen me- tabolism by comparison to 15O-PET. Conducting these studies on a PET/MR system will ensure the PET and MRI measurements are collected under the same physiological state. The MRI oximetry method will be combined with FDG PET to investigate the effects of a ketogenic supplement on oxygen and glucose metabolism in AD patients. The outcomes of this project will include validating an MRI-based method for imaging oxygen metabo- lism and demonstrating how it can be combined with FDG PET to evaluate a treatment that targets the brain energy gap associated with AD. Replacing 15O-PET by MRI oximetry would greatly enhance the feasibility of imaging substrate (i.e. glucose) and total energy metabolism (i.e. oxygen) considering how few PET sites with 15O capabilities remain, while FDG is the most used tracer. The longer-term goals are to provide non-invasive, neuro-imaging tools for studying individuals with ADRD and evaluate effectiveness of emerging drugs.

NIH Research Projects · FY 2026 · 2024-03

Abstract. SHIV-infected rhesus macaques (RMs) develop HIV-1 broadly neutralizing antibodies (bNAbs) via Env-Ab coevolutionary pathways that recapitulate events in HIV-1 infected humans (Science 371:eabd2638, 2021). We hypothesize that SHIV-infected RMs can thus serve as a novel “molecular guide” for HIV-1 immunogen design, allowing for an iterative and reproducible analysis of the critical steps in bNAb elicitation, including efficient priming of naïve B cell precursors, immunofocused boosting, and affinity-maturation leading to breadth and potency. In this application submitted in response to PAR-23-169 (Innovation for HIV Vaccine Discovery), we propose to design novel germline-targeting SHIVs and to use them to decipher each of these steps in SHIV-infected monkeys where, unlike in humans, we can analyze multiple animals infected by the same SHIVs and identify Env-Ab co-evolutionary pathways that are shared among different animals. Based on these findings, we will design and test novel prime and boost protein immunogens corresponding to key Env “immunotypes” responsible for elicitation of bNAbs in the SHIV-infected monkeys. Our proposal focuses on immunogen design for one of the most advanced HIV-1 vaccine targets (fusion peptide, FP) but one where boosting regimens that lead to high level breadth and potency have been challenging to develop. Our immunogen platforms include BG505, CH1012 and Ce1176 transmitted/founder Envs, each of which we show to be capable of eliciting FP bNAbs in RMs. The novel aspect of this application is that we propose to enhance the efficiency of priming, boosting and affinity maturation steps by designing new SHIVs with selected glycans deleted surrounding the FP site of vulnerability. We show that such SHIVs replicate efficiently and immunofocus early B cell responses to FP epitopes leading to neutralization breadth and potency. By deciphering Env-Ab coevolution leading to consistent bNAb induction in SHIV infection, we will then design and test new FP targeted protein immunogens that are practical for vaccine development. Specific aims are: (i) to construct novel SHIVs with BG505, CH1012 and Ce1176 Envs that are depleted of N-linked glycan sites surrounding the FP and to characterize their antigenicity, neutralization sensitivity, infectivity and replication in primary rhesus CD4+ T cells; (ii) to infect RMs with glycan-depleted SHIVs from Aim #1, epitope-map autologous and heterologous neutralizing antibody responses, identify germline B cell precursors to FP bNAbs and decipher molecular patterns of Env-Ab coevolution leading to neutralization breadth; (iii) to design novel Env trimers corresponding to key Env immunotypes that bind FP bNAb UCAs and intermediate stage Abs and to express them as stabilized prefusion Env trimer immunogens; and (iv) to conduct a proof-of-concept preclinical vaccine trial in RMs testing the hypothesis that SHIV-guided, B lineage-designed Env trimers can prime, boost and affinity-mature bNAb responses in RMs to an extent that is superior to conventional immunogens and that these bNAbs protect RMs against low-dose intrarectal challenge by heterologous tier-2 SHIVs.

NIH Research Projects · FY 2026 · 2024-03

Project Summary In sexually reproducing organisms, meiosis reduces the chromosome complement by half to generate haploid gametes. During meiosis, homologous chromosomes undergo pairing, synapsis, recombination, and faithful segregation. As such, defects in meiosis are a leading cause of both infertility and birth defects in humans. While a large number of meiosis-specific factors are involved, ubiquitously expressed factors also play critical roles. The meiotic process is generally conserved from single-cell eukaryotes to multicellular metazoans; however, species-specific meiosis factors and functions have evolved. Our genomic and proteomic screens have identified a large number of novel mammalian meiosis factors and we plan to determine the molecular function of a number of key factors in the regulation of meiosis in both sexes. Meiotic recombination is essential for genome integrity in gametes and critical for genome evolution by increasing genetic diversity at the population level. Meiotic recombination begins with formation of meiotic DNA double strand breaks (DSBs). There are major knowledge gaps in our understanding of this meiotic process. One main challenge is to understand the mechanism that prevents meiotic DSB formation from getting out of control. Only a subset of the meiotic DSBs are processed into crossovers by a large protein network. What is the molecular mechanism underlining the crosstalk in this protein network? What additional proteins are involved in the processing of DSBs into crossovers? In many species, chromosomal synapsis is tightly coupled with meiotic recombination. However, the process of synapsis initiation and maintenance remains poorly understood. These challenges will be tackled in this application through deciphering novel molecular networks underlying regulation of mammalian meiosis. Our innovative combination of genetic, genomic, proteomic, cell/molecular biological, and biochemical approaches will focus on the following critical but challenging aspects of meiosis: initiation of meiotic DNA double strand breaks, regulation of meiotic recombination, initiation and maintenance of chromosomal synapsis, and origin of sex chromosome aneuploidy. Completion of the proposed studies will provide novel mechanistic insights into key meiotic processes, identify new candidate fertility factors, and elucidate the molecular etiology of sex chromosome aneuploidy.

NIH Research Projects · FY 2025 · 2024-03

Abstract Digital assays — in which ultra-sensitive molecular measurements are made by performing millions of parallel experiments in picoliter droplets — have generated much recent enthusiasm due to their single molecule resolution of RNA, DNA, and proteins, and their robustness to reaction conditions. These assays have enormous potential for the diagnosis of difficult to diagnose diseases, such as pancreatic cancer, but are currently confined to laboratory settings due to the cumbersome instrumentation necessary to generate, control, and measure tens of millions of independent droplets. To overcome this challenge, we are developing a hybrid microelectronic / microfluidic chip to ‘unlock’ droplet-based assays for clinical use. Our microdroplet megascale detector (µMD) can generate and detect the fluorescence of millions of droplets per second (1000× faster than existing digital approaches), while achieving a 1000x greater sensitivity than conventional ELISA or ddPCR, using only a conventional cell phone camera. The key innovation of our approach is borrowed from the telecommunications industry, wherein we modulate the excitation light with a pseudorandom sequence that enables individual droplets to be resolved that would otherwise overlap due to the limited frame rate of digital cameras. Building on the success of our R21, we propose to develop a platform technology to ultrasensitively quantify proteins, ctDNA, and single EVs in an integrated device, directly in patient blood, to address critical issues in multimodal diagnostics. In collaboration with the Abramson Cancer Center and building on prior work together on multi-modal diagnostics, we focus our attention on Pancreatic ductal adenocarcinoma-the third leading cause of cancer- related death in the United States with an overall 5-year survival of only 9%.