Johns Hopkins University

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY / ABSTRACT Sarcoidosis is a multi-organ, immune-mediated inflammatory disease with a highly variable clinical presentation and disease course. Additionally, the mortality rate from sarcoidosis appears to be rising over the past decades. A significant challenge in the management of sarcoidosis is the lack of reliable diagnostic and prognostic biomarkers. The objective of this project is to define microRNAs (miRNAs) that can be used as diagnostic biomarkers and to associate miRNA expression with clinical characteristics in sarcoidosis for the development of prognostic biomarkers. A genomic approach to biomarker development is well suited for complex, polygenetic diseases such as sarcoidosis and will be the focus of this project. miRNAs have significant potential as biomarkers in sarcoidosis given their role as epigenetic modifiers and immune response regulators. Prior to this grant, we performed miRNA sequencing on bronchoalveolar lavage (BAL) cell samples in a discovery cohort comprised of sarcoidosis cases and healthy controls; this process identified 7 differentially expressed miRNAs, which we will use in all the aims of this current proposal. In Aim 1, we will validate the 7 differentially expressed miRNAs in a validation cohort comprised of cases, healthy controls, and interstitial lung disease controls. The miRNAs that are validated will then be tested as a diagnostic biomarker for sarcoidosis. In Aim 2, we determine if the expression levels of the 7 differentially expressed miRNAs at the time of BAL reflect clinical manifestations of sarcoidosis. We will also determine if the miRNA expression levels at the time of BAL can predict changes in clinical manifestations over two years. Finally, in Aim 3, we will characterize the stability of the 7 differentially expressed miRNAs over two years in a subset of sarcoidosis cases. This finding, along with the results from Aim 2, will help in the development of prognostic biomarkers in a future grant. This proposal will provide a strong foundation for Dr. Lin’s long-term goal of becoming an independently funded physician-scientist in the field of translational sarcoidosis and environmental/occupational pulmonary research. Specifically, this proposal will continue to build her expertise in genomic biomarker development, longitudinal study design, and statistical analyses specific to sarcoidosis research. This proposal will also generate preliminary data for her future R01 grant submissions.

NIH Research Projects · FY 2026 · 2023-05

Chemotaxis occurs during a number of key physiological events including angiogenesis, embryonic development and wound healing. It also contributes to disease progression in pathological conditions such as cancer metastasis and arthritis. The goal of the current proposal is to reveal how biochemical reactions and physical characteristics, such as membrane curvature, deformation, and assembly phase, interact with one another in achieving dynamic, accurate yet highly efficient cell migration. Chemotaxis has been understood mainly in the perspective of signal transduction, while if and how physical properties of membranes play a role, and how they interact with signal transduction remain largely unknown. By newly developing and implementing a series of molecular actuators that can directly probe membrane properties with high spatio-temporal precision inside lively migrating cells, we will reveal an interplay between signal transduction and membrane mechanics. What molecular mechanisms generate local membrane curvatures developing into filopodia and lamellipodia? In sensing chemoattractants, cells polarize by undergoing asymmetric membrane deformation consisting of filopodia and lamellipodia at the front, and membrane retraction at the rear. We recently found that curvature-sensitive proteins are a missing link between actin cytoskeleton and membranes. The result made us hypothesize that actin machinery and curvature sensing and remodeling proteins, when properly modulated in a feedback loop, are sufficient to produce desired types of membrane deformations such as lamellipodia and filopodia. We will thus identify a particular combination of Rho GTPases, actin regulators, and BAR proteins, and the molecular logic thereof, that are responsible for formation of filopodia and lamellipodia. How do signaling components in migrating cells respond to membrane deformation? Migrating cells exhibit dynamic morphological changes at plasma membranes and nuclear envelopes “as a consequence” of cytoskeletal rearrangement regulated by signal components. To explore a possibility that membrane deformation talks back to cytoskeletal and signal components, we will deploy molecular actuators that can directly deform membranes. We will then quantify subsequently emerging activity of signaling components such as receptor tyrosine kinases, PI3K, and small GTPases, as well as transcription factors such as YAP and Elk. How does the phase-separated cytoskeletal biomolecular condensate play a role in membrane deformation? Actin networks can undergo formation of biomolecular condensates at the plasma membrane due to weak multivalent interactions among actin regulators. To examine the physiological importance of such phase separation events, we will adapt molecular techniques to assemble or disassemble the condensates. These operations will uniquely achieve gain- or loss-of function manipulations without altering an amount of the molecular constituents; what is altered is their physical assembly status. We will characterize cell migration phenotypes before and after deploying phase manipulations.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Breast cancer is a widespread disease that will claim the lives of over 40,000 US women in 2022 alone, with the majority of these deaths resulting from stage IV disease, in which cancer has metastasized to distant regions, as metastatic disease is difficult to treat and has few effective treatment options. Breast cancer cells metastasize by invading the basement membrane, invading into blood vessels, circulating to distant tissues, leaving the blood vessel, and colonizing that tissue. The epithelial cell adhesion molecule (EpCAM) plays a role in cell adhesion, migration, and invasion, and has been linked to several epithelial cancers, including breast, prostate, and colorectal cancer. Despite its potential to facilitate several steps of the metastatic cascade and its link to epithelial cancers, the specific role of EpCAM in metastasis remains unknown. In his study of normal tissues, primary tumors, and metastatic nodules of 17 metastatic breast cancer patients, our collaborator Dr. Pedram Argani determined that EpCAM protein expression was increased in metastatic nodules, without a corresponding increase in EpCAM mRNA expression. We therefore hypothesize that post-translational modification of EpCAM, which has three known N-glycosylation sites and no known O-glycosylation sites, stabilizes and increases the abundance of EpCAM in metastatic tissues. Our recent study of these same samples found that the abundance of N-glycans was significantly increased in metastatic tissues versus primary tumors versus normal tissues and identified 25 significantly differentially abundant N-glycans of interest. Our study also found that EpCAM protein expression was significantly increased in metastatic tissues compared to primary tumors and was significantly statistically correlated with the expression of seven N-glycans. Taken together, these data suggest that EpCAM N-glycosylation may play a role in metastatic progression. The central hypothesis of this project is that increases and changes in N-glycosylation stabilize EpCAM and allow it to facilitate metastasis. I propose to test this hypothesis through the following specific aims: Aim 1: To elucidate the impact of EpCAM N-glycosylation on EpCAM stability and adhesion in human breast cancer cells; Aim 2: To assess the impact of EpCAM N- glycosylation on metastasis in mouse models of breast cancer. These aims will be achieved through a combination of cancer biology, biochemistry, and mass spectrometry approaches in human triple-negative breast cancer cell lines and breast cancer xenograft models. This work is significant because it will elucidate the role of N-glycosylation of EpCAM in metastatic breast cancer, determine the specific N-glycosylation profile of EpCAM in metastasizing breast cancer cells, and may subsequently uncover novel therapeutic targets and strategies in metastatic breast cancer.

NIH Research Projects · FY 2025 · 2023-05

Project Summary Alzheimer's Disease (AD) is a neurodegenerative disease affecting millions of people around the globe. AD pathology includes neuronal cell death, synaptic dysfunction, and corresponding behavioral deficits. However, the molecular basis underlying AD pathologies remains unclear. Glycerophosphodiester phosphodiesterase 2 (GDE2) is a member of a six-transmembrane protein family that releases glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. In the developing nervous system, GDE2 promotes neuronal differentiation in the central nervous system (CNS) using its GPI-anchor cleavage mechanism. During aging the loss of GDE2 leads to AD-like phenotypes, including Aβ production, synaptic protein loss, and neurodegeneration. In human AD brain tissue, GDE2 aberrantly accumulates intracellularly compared to healthy control brains, suggesting that GDE2 dysfunction could contribute to AD pathophysiology. GDE2 mediates its functions in regulating Aβ production and synaptic protein loss via regulation of surface expression of the GPI-anchored protein RECK (reversion-inducing cysteine-rich protein with Kazal motifs). RECK is a potent inhibitor of metalloproteases and is recently implicated in canonical Wnt signaling. Increased expression of membrane RECK leads to a decrease in synaptic proteins that is independent of Aβ production and levels of membrane RECK are highly elevated in AD patient brain. The guiding hypothesis of this proposal is that GDE2 regulates synaptic function through the cleavage of RECK and that dysfunction of this pathway contributes to AD synaptic pathology and cognitive changes. I will test this hypothesis through a multi-pronged approach that will utilize electrophysiological, behavioral, and molecular approaches. In Aim 1, I will assess the requirement for GDE2 in pre- and post-synaptic function in mice. In Aim 2, I will perform a variety of behavioral experiments testing cognition to define corresponding behavioral abnormalities in mice lacking GDE2. Finally, in Aim 3, I will probe the molecular mechanism through which GDE2 and RECK act to affect synaptic biology. These experiments will expand our knowledge of the role GDE2 plays in synaptic biology and could potentially help us better understand the mechanistic basis of AD pathologies. This research will be performed in a highly collaborative environment, where I will have numerous opportunities to receive quality mentorship and training, to develop my written and presentation skills, and to grow as a mentor and teacher to more junior scientists. Overall, the Kirschstein-NRSA grant will support both my research aimed at discovering the synaptic biology underlying neurodegeneration and my development as an independent scientist.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY In low oxygen conditions, such as the hypoxic microenvironment of solid tumors, cells cannot perform mitochondrial respiration and rely solely on glycolysis for ATP generation. Thus, to overcome this deficit in energy production, cells require mechanisms to enhance ATP generation in response to hypoxia. We discovered that in hypoxic yeast cells, the enzymes of the glycolysis pathway, which are diffusely localized in the cytosol under normoxic conditions, organize into non-membrane-bound structures we term Glycolytic (G) bodies. G bodies constitute ribonucleoprotein (RNP) condensates formed by phase separation. G body formation is correlated with increased glucose consumption and cell survival and proliferation under hypoxia. Similar structures have been observed in C. elegans and human cancer cell lines, supporting G body formation as an evolutionarily conserved adaptive response. We hypothesize that G bodies enhance rates of glycolysis by coordinating the multiple steps of the pathway to promote cell survival during hypoxic stress conditions. Using biochemical purification, we have determined the protein and RNA constituents of G bodies and our genome-wide deletion screen identified key signaling pathways that influence G body formation. A major gap in the condensate field is the lack of direct evidence for condensate function. Our preliminary results indicate that G bodies exhibit enhanced glycolytic enzyme activity, thus supporting a functional role for these novel RNP condensates. In this proposal, we will employ a diverse array of experimental strategies to investigate G body activity, physiological impact, biogenesis, biophysical properties, and functional conservation. Mechanisms by which G bodies potentiate glycolytic enzyme activity will be pursued using our purified G body in vitro system and by utilizing novel metabolic biosensors in vivo. We will also determine the global metabolic impact of G bodies through metabolomic approaches and investigate the genetic regulation of G body biogenesis and function mediated by conserved energy-sensing signaling pathways. Molecular and biophysical analysis of G bodies will examine the role of non-canonical RNA binding by glycolysis enzymes in condensate formation via phase separation. Finally, we will take the lessons learned in yeast and apply them to human cancer cell lines and 3D spheroid cultures to study the conservation of G body biophysical properties, biogenesis, and physiological function. Successful outcomes of our research will reveal novel basic principles of RNP condensate regulation and function across species and provide mechanistic insights and potential therapeutic strategies to mitigate hypoxic adaption and cell proliferation in solid tumor microenvironments.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ ABSTRACT Kidney transplantation (KT) is a growing treatment for older adults with end-stage renal disease (ESRD), but there is vast heterogeneity in KT outcomes. Older adults are more likely to be listed as inactive (on the waitlist but ineligible for KT), which is associated with increased waitlist mortality and worse post-surgical outcomes. Those awaiting KT also experience depressive symptoms, pain, loss of physical function, and social isolation, which can contribute to waitlist mortality and decrease chances of KT. As of April 2022, 94,249 people were awaiting KT with an estimated 44% currently inactive. There is a critical need for enhanced models of care to improve inactive waitlist outcomes. The purpose of this study is to adapt and pilot test the evidence-based Community Aging in Place- Advancing Better Living for Elders (CAPABLE) intervention to address barriers for KT waitlist activation which involve symptom burden, self-management, social support, health literacy, patient activation and home function. CAPABLE equips older adults to age in their homes using person-directed priorities and a strengths-based tailored approach by a nurse, occupational therapist and handy worker (PI: Szanton, co-primary mentor). CAPABLE improves function, pain, depressive symptoms, and quality of life while decreasing hospitalizations and nursing home admissions. CAPABLE also improves healthcare engagement and self-efficacy which are key components to remaining active on the KT waitlist. Our adapted CAPABLE–Transplant model will extend services to include options for internet access, training, patient portal usage and patient-directed online social engagement to address the noted isolation. We hypothesize that decreasing patient and clinician reported barriers will decrease time inactive on the KT waitlist status. We plan to examine CAPABLE-Transplant among those with inactive KT waitlist status in a two-phase developmental study leveraging partnership with the JHU Comprehensive Transplant Center and an ongoing, prospective NIA R01-funded cohort study of individuals awaiting KT for recruitment (PI: McAdams-DeMarco, co-primary mentor) through the following aims: (1) To develop an adaptation of CAPABLE targeting those currently KT inactive, (2) To iteratively refine the CAPABLE -Transplant prototype for those currently KT inactive and, (3) To pilot test the CAPABLE-Transplant intervention in a 30 person 1:1 randomized waitlist control trial delivered over 16 weeks with outcomes (e.g. waitlist status, symptom burden, social networks) evaluated at 0,16, and 32 weeks post-randomization to test feasibility, acceptability, fidelity of CAPABLE-Transplant and estimate preliminary effects sizes for a future efficacy trial. To our knowledge, there are no other home-based programs that address patient-directed goals and the home environment among those inactive awaiting KT. This work will form the basis for a future R01 to expand to other KT centers and/or into other transplant populations conducting a larger community-based, efficacy trial.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Our research program is directed towards innovating and advancing optical tools for the visualization and quantification of latent biomolecular processes across multiple levels of biological organization. The MIRA project will support and improve our analytical toolkit, which spans from surface-enhanced Raman spectroscopic (SERS)-based molecular imaging probes to self-actuating single-cell analysis platforms and biomimetic structures for cellular mechanotyping. Crucially, with support from the MIRA proposal, we will develop three new, complementary platforms to address pressing questions in multiplexed molecular analysis, intracellular magnetic sensing, and targeted imaging of cells and tissues. First, we plan to realize a novel Raman spectroscopic sensing method by fusing SERS with coherent vibro- polariton interactions in the strong coupling regime. While highly desirable, achieving vibrational strong coupling (VSC) between ground-state molecular vibrations and an optical cavity has remained elusive. Combining SERS nanoprobes with rationally designed Fabry-Perot cavities, we present a practical scheme to render VSC that would simultaneously enhance the strength of Raman scattering and enrich its spectral features paving the way for ultrasensitive and highly multiplexed analyte detection. Second, we aim to develop an ultrasensitive nanoscale magnetometer to probe spin effects in biomolecules, an important but poorly understood quantum effect in biological systems. We will implement a DNA-assisted self-assembly approach to pair nitrogen vacancy-center in nanodiamond (NVnD) with plasmonic nanocavities. The accompanying enhancements in NVnD sensitivity and spatiotemporal resolution will permit the detection of currently undetectable ion flux-induced weak magnetic fields (WMF) and to examine the role of WMF in affecting the spin dynamics of cryptochrome-generated radical pairs. Third, we seek to harness biocompatible click condensation reactions to create a new class of synthetic peptide-based Raman imaging nanoprobes involving enzyme-regulated intracellular self-assembly. Our nanoprobes offer multiple advantages for targeted cellular imaging: higher accumulation and reduced efflux due to in situ probe assembly; high sensitivity due to the presence of repetitive units of a π-conjugated functional group in a single structure; and exquisite specificity owing to the easily distinguishable vibrational mode in the cell silent spectral region.

NIH Research Projects · FY 2025 · 2023-05

SUMMARY Auditory verbal hallucinations (AVH) afflict more than 80% of schizophrenia (SZ) patients and are treatment resistant up to 40-45%. AVHs increase the risk of suicidal, aggressive behavior, reduced work attainment and impairment in specific cognitive domains. Considering that 25-50% of persons with SZ are treatment-resistant (TR-SZ), there is unmet public health need to treat persistent AVH in TR-SZ. SZ is a disorder with disruptions within corticostriatothalamic circuits ((CST) thus potentially amenable to modulation via Deep Brain Stimulation (DBS). DBS-SZ may hypothetically modulate AVH through its effect on projections from superior temporal gyrus (STG) to basal ganglia (BG), via the substantia nigra pars reticulata (SNr) and mediodorsal nucleus of the thalamus (MDN). There is evidence that the SNr-MDN-STG loop is dysfunctional in SZ and lesions within this loop cause new onset SZ-like hallucinations. Modulation of the SNr-MDN-STG loop could result in treatment of persistent hallucinations in SZ. In support of this hypothesis, we have preliminary evidence that bilateral SNr DBS induces sustained remission of chronic AVH in a TR-SZ patient and their improvement in a second patient. We hypothesize (Aim 1) that SNr DBS decreases AVH measured by in-clinic BPRS, and at-home-Ecological momentary assessment technique by at least 20% without serious adverse events when comparing baseline to 60 weeks DBS stimulation. For more granular examination of DBS effect on AVH, we will use in-clinic Auditory Vocal Hallucinations Rating Scale (AVHRS), and the Scale for the Assessment of Positive Symptoms (SAPS) (Aim 1). A potential neural mechanism of SNr DBS modulation for AVH might be restoring oscillatory activity in the SNr-MDN-STG loop. Abnormalities in γ oscillations (30–100 Hz) of the electroencephalogram are ubiquitous in SZ. SNr recordings in persons with SZ is uncharted, but low-γ oscillations have been recorded from the SNr in ketamine- treated rats, a well-established SZ model--these are hypothesized to reflect cognitive and sensory, rather than motor impairment. We hypothesize that AVHs in SZ are associated with excessive gamma power in the SNr, and that these abnormal SNr gamma oscillations can be targeted with bilateral SNr DBS resulting in reduced AVH. We will collect home LFP recordings to assess changes in SNr LFP gamma power in DBS on vs off and explore if DBS-related oscillatory changes correlate with AVH changes (Aim 2). Since AVH are associated with impairments in saccadic eye-movements s, we will also explore if DBS-related oscillatory changes correlate with saccadic impairments and their improvements Aim 2.)

NIH Research Projects · FY 2026 · 2023-05

Project Summary Advances in eukaryotic gene expression have provided a comprehensive list of transcription-related proteins, their biochemical activities and structure-function relationships, and revealed the importance of histone modifications and nucleosome remodeling enzymes that cooperate with sequence-specific DNA binding transcription factors, resulting in a transcriptionally poised chromatin architecture at gene promoters and enhancers. However, major challenges remain in the lack of knowledge of the timescales and kinetics by which epigenetic and transcription proteins operate on chromatin substrates. This proposal will address these challenges by focusing on quantitative kinetics of chromatin remodeling and transcription, using state-of-the-art single-molecule imaging techniques applied to living cells and immobilized chromatin templates in vitro, combined with traditional biochemistry and yeast molecular genetics. Anticipated findings are the identification of key reaction intermediates, order of events, and rate-limiting steps that will dramatically advance basic, mechanistic understanding of transcription on native chromatin with high impact on other areas of eukaryotic DNA metabolism.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT: Recent clinical and preclinical evidence has shown that gliomas can initially grow, invade, evade antiangiogenic therapies and eventually recur by hijacking or “co-opting” the brain’s preexisting blood vessels. Vascular co-option is a nonangiogenic glioma growth mechanism in which, co-opting tumor cells cause astrocytes to lose intimate contact with blood vessels, i.e. cause gliovascular uncoupling (GVU), and alter cerebral hemodynamics. Additionally, in aggressive high- grade gliomas (e.g. glioblastoma or GBM), vessel co-option facilitates the migration and invasion of cancer cells into healthy brain tissue. Yet, the evolution of vessel co-option over the glioma’s life-time, resultant GVU and hemodynamic changes remain poorly understood due to a lack of microvascular-resolution lifecycle and multimodality/multiscale imaging approaches. Moreover, co-optive glioma growth is radiologically undetectable due to an absence of contrast enhancement and the lack of specificity of conventional MRI (e.g. T2/FLAIR) approaches. Therefore, our goal is to use an image-based systems biology approach to elucidate the hemodynamics of co-optive glioma over its lifecycle and develop an fMRI biomarker of co-option induced GVU. Guided by compelling preliminary data, we will pursue three Specific Aims: (1) Characterize vessel co-option in a patient-derived glioma xenograft (PDX) over its lifecycle with multiscale imaging; (2) Develop an image-based model of brain-wide hemodynamic changes induced by vessel co-option in glioma; and (3) Determine if rs-fMRI can detect vessel co-option induced GVU in a patient-derived glioma xenograft. Under Aim1, we propose a paradigm-shifting approach that employs a miniscope for microvessel resolution (~5 µm) multicontrast in vivo imaging of co-option induced hemodynamic changes over the lifecycle of a patient-derived glioma xenograft. We will complement these microvascular-scale measurements with multimodality/multiscale whole-brain data from ex vivo CT/MRI/light sheet microscopy (LSM) in the same animal to corelate structural/functional/cellular changes in the vascular microenvironment (VME). Under Aim2, we employ these data in a model of co-option induced hemodynamic dysregulation to simulate brain-wide changes that could be exploited as fMRI biomarkers of co-optive glioma. Under Aim3, we will determine if resting-state fMRI (rs-fMRI) can detect GVU in a co-optive PDX and differentiate it from non-co-optive glioma growth. Our approach is innovative because it blends cutting-edge advances in miniaturized microscopy, multiscale/multimodality imaging and image-based systems biology. The proposed research is significant because these studies will establish: (i) freely downloadable, co-registered multiscale data for cancer systems biology investigators; (ii) a hemodynamic model for co-optive glioma; (iii) a novel biomarker of glioma co-option with the potential to transform patient management and stimulate the development of therapies to thwart antiangiogenic resistance. We also expect this approach to be adaptable to other CNS diseases dependent on vessel co-option (e.g. brain metastases).

NIH Research Projects · FY 2026 · 2023-04

Project Summary High-risk myelodysplastic syndrome (MDS) arises from malignant primitive cells that are resistant to current therapies. These cells exhibit a growth benefit in the MDS microenvironment and their elimination is challenging due to the lack of targetable markers distinguishing them from healthy primitive cells. We found that men with MDS and myeloproliferative neoplasms (MPN) have worse survival and higher disease burden in their primitive cells compared to women. To shed light into these sex differences, we evaluated the expression of known androgen receptor (AR) target genes in MDS/MPN samples. We found that one of the top AR-regulated genes, CCRL2, is overexpressed in primitive cells from MDS patients. CCRL2 is an atypical chemokine receptor as it lacks G-protein binding domain, is internalized via endocytosis and its C-terminal region is required for its surface localization. Our published results showed that CCRL2 induces MDS growth and activates JAK2 highlighting this receptor as a potential target in MDS. CCRL2 knockdown suppresses growth-related pathways, most prominently transferrin receptor (TFRC) and E2F targets. However, as CCRL2 lacks signaling domains, the exact mechanism is unclear. We hypothesized that CCRL2 interacts with TFRC in early endosomes and activates it while CDK/E2F is regulated by CCRL2 via JAK2. We also hypothesize that CCRL2 C-terminal region is essential for its oncogenic properties. We found that CCRL2 deletion sensitizes MDS cells to azacitidine, the most commonly used MDS therapy. Inhibition of JAK2, a CCRL2 target increases azacitidine efficacy in CCRL2 wild- type but not in CCRL2 knockdown cells. Thus, JAK2 may mediate the CCRL2-regulated azacitidine resistance. This also suggests that targeting other CCRL2-regulated pathways can be selectively effective against CCRL2- expressing cells. Thus, we hypothesize that inhibition of CCRL2-regulated pathways or agents suppressing CCRL2 levels are toxic against CCRL2-expressing cells and can increase azacitidine efficacy. In Aim 1 we will confirm the induction of TFRC growth-related activity by CCRL2, define JAK2 role in CCRL2-mediated growth pathways regulation and determine the involvement of CCRL2 C-terminal region in its growth effects by performing immunofluorescence, proteomics, and CCRL2 gene editing. In Aim 2 we will confirm the implication of JAK2/STAT in the CCRL2-mediated azacitidine resistance and discover other agents with selective efficacy against CCRL2-expressing cells by performing drug and CRISPR-Cas9 knockout screens. Selected agents will be combined with azacitidine in an inducible-CCRL2 MDS xenograft model. These studies will provide the PI the opportunity to gain critical skills and expertise to study the molecular biology of MDS growth with emphasis in the role of CCRL2 and discover novel targeted therapies for this disease. These objectives will be accomplished through formal coursework, scientific programing, and direct mentorship by experts in molecular genomics, pharmacology and computational biology. The proposed career development plan and research aims will provide a pathway to a career as an independent investigator studying MDS/MPN molecular biology.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY The Johns Hopkins Center for American Indian Health and its partners, the White Mountain Apache Tribe and Navajo Nation are uniquely positioned and prepared to advance suicide prevention science through the funding opportunity “Service-Ready Tools for Identification, Prevention and Treatment of Individuals at Risk for Suicide” (RFA-MH-21-110). Native American populations experience substantially higher rates of suicide and the sharpest increases over time of any racial group. These disparities are the legacy of colonization, attempted genocide, historical trauma, and ongoing injustices, including chronic underfunding of health care and mental health services. Despite these adversities, NAs have exercised tribal sovereignty to support some of the most innovative, practical, and effective approaches to suicide prevention. One such model, the Celebrating Life program (CL), a community-based suicide surveillance and case management system, developed originally by the White Mountain Apache Tribe (WMAT) and shown to help contribute to reductions in suicide attempts and deaths over time. The CL program is now being scaled through the Southwest Hub for Youth Suicide Prevention (U19MH113136). As this program has scaled, our pilot work has revealed barriers to implementation, including 1) challenges to risk identification, and 2) difficulties matching individuals’ level of risk to evidence-based interventions as a means to improve scarce resource efficiencies. To help overcome these barriers to implementation, NIMH funded our team to develop and pilot test NATIVE-RISE (U19MH113136-02S3). NATIVE-RISE is a systems-level strategy that leverages predictive analytics to enhance risk identification and integrates and improves the efficiency of local NA case managers who deliver brief contact interventions. The proposed project will leverage our team’s historic MOU with Indian Health Service (IHS) and decades-long trust-relationships with tribal partners to test NATIVE-RISE and its integration in partnership with three NA-serving health care settings through a Hybrid Type III stepped-wedge cluster randomized implementation trial. Specifically, this project aims to 1) optimize the implementation of NATIVE- RISE across three NA-serving health care settings, 2) determine the effectiveness of NATIVE-RISE at improving the reach of evidence-based suicide prevention services, and 3) describe the costs associated with implementing NATIVE-RISE to inform scale-up and sustainability. Our application address key priority populations for NIMH (NOT-MH-21-090) and is in line with NIMH’s strategic plan Objective 4.3. If results are achieved, NATIVE-RISE will become an effective and scalable approach that improves services to prevent suicide among NA populations and advances our understanding of how to implement predictive analytics and more personalized care for suicide prevention.

NIH Research Projects · FY 2025 · 2023-04

Project Summary/Abstract Nearly 2 million women pass through U.S. jails each year, with nearly 8,000 admissions of pregnant people with opioid use disorder (OUD). Providing evidence-based treatment to these individuals is essential to addressing the opioid epidemic, to optimizing maternal and newborn outcomes, and to promoting maternal health equity. There is increasing recognition of the critical role of jails in providing access to medication treatment for opioid use disorder (MOUD) as a means of curbing the opioid epidemic, reducing overdose deaths, and promoting racial equity for a group of individuals who are systematically marginalized. While there is a growing number of jails that are expanding access to MOUD, strategies that are specific to the unique needs of pregnant people are lacking. Moreover, many jails still do not provide MOUD even in pregnancy, despite the well-established standard of care of avoiding withdrawal and the known benefits of MOUD specifically for pregnant individuals. Our long-term goal is to ensure that pregnant people with OUD in jails receive appropriate and timely care to optimize their long-term wellbeing and that of their infants. The overall objective is to develop and pilot an adaptable implementation strategy and toolkit for jails to be able to provide access to pregnancy-specific OUD care. The rationale is that jails vary tremendously in size, resources, and health care delivery systems, and need tools they can tailor to their environment. Ensuring jails provide access to MOUD, and in ways that are tailored to the distinctive medical, mental health and social structural aspects of care for pregnant people with OUD is essential for improving short and long-term pregnancy, recovery, and intergenerational outcomes. This project will engage multiple stakeholders, including directly impacted people, to design then pilot a patient-centered and jail-feasible implementation strategy that will facilitate and enhance jails’ implementation of MOUD for pregnant people. The strategy will contain a menu of tools to assist jails with immediate needs to provide MOUD to pregnant people entering jails; with pregnancy-tailored counseling; and with other support services and linkages to care that center the obstetrical, psychosocial, and structural needs of this population. The implementation strategy will also be adaptable to a variety of types and geographies of jails. They will then be piloted at four jails with different baseline capacities and services for MOUD for pregnant people. The steps proposed in this R34 are necessary for building the tools, outcome measures, and capacity that will be scaled-up in a future R01 hybrid implementation/effectiveness trial to improve OUD treatment access and maternal and infant health outcomes for this long-overlooked group.

NIH Research Projects · FY 2026 · 2023-04

There is a consensus that environmental pollutants are a risk factor for Alzheimer's Disease Related Dementias (ADRD). Emerging evidence has shown that environmental stressors (e.g., urban and roadside air pollution) contribute to dementia. Our supporting epidemiological results have determined that annual mean particulate matter (PM) pollution in the USA is significantly associated with an increased risk of first hospital admission with ADRD. We found strong evidence of linearity in concentration-response relationship at PM concentration less than 16 μg/m3 (95th percentile of the PM distribution). PM pollutants can target the central nervous system (CNS). However, what specific PM pollutants are associated with dementia and the underlying mechanisms are poorly known. One of the most common dementia is called “Lewy Body Dementia (LBD)” with the typical hallmark of αS pathology, including Dementia with Lewy body (DLB) and Parkinson’s Disease with Dementia (PDD). Patients with LBD suffer from cognition and memory dysfunction, behavioral and mood symptoms (e.g., depression, anxiety, and etc.), affecting 1.4 million individuals and their families in the USA. Furthermore, 30% of the Alzheimer’s disease (AD) subjects with αS pathology generally exhibit a more rapid rate of cognitive decline than subjects with AD alone. Substantial postmortem studies by Braak et al. showed the presence of αS pathology was initialized in the olfactory bulb (OB) and gastrointestinal tract, and spread to the brain following stereotypical anatomical stages, resulting in autonomic, neuropsychiatric, and cognitive dysfunction. In addition to clinical observations, emerging evidence has shown pathogenic αS spreading is a master trigger to cognitive impairment (CI) by using inoculation of recombinant αS preformed fibrils (PFF). The majority cases of αS-related dementia (LBD and ⅓ AD) are sporadic and have many causes. Braak’s theory well supports the hypothesis that αS-related dementia may begin when foreign stressor (e.g., PM pollutants) enter the body via the nose/gut, induce αS aggregation and subsequent prion-like pathology spreading into the CNS, which results in ADRD. While epidemiological studies demonstrating an association of ADRD with air pollutants are relatively abundant, there is a clear unmet need for more mechanistic research. This knowledge is critical for achieving a complete understanding of the etiology of LBD and the translation of such knowledge to novel prevention and treatment strategies. This is especially important for understanding the causes of ADRD, and importantly, environmental toxicant risk factors are potentially modifiable.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Tuberculosis is the bacterial infection that kills the most people worldwide, especially in India, which has the highest burden in of multidrug-resistant tuberculosis (MDR-TB) in the world. Delays in adequate treatment due to slow diagnostic tests and the usual one-size-fits-all MDR-TB treatment strategy lead to additional drug resistance, terrible treatment-associated side effects, and high mortality. Rapid next generation sequencing (NGS) from uncultured samples is a novel diagnostic tool that can predict resistance and minimum inhibitory concentration (MIC) ranges for MDR-TB within 2 days of presentation–weeks before culture-based susceptibility tests provide results. NGS-predictions allow providers to tailor MDR-TB treatment and choose doses to target specific drug levels at the start of treatment. Most MDR-TB drugs reach steady state within 2 weeks, so early therapeutic drug monitoring (TDM) supported by direct observation of therapy could verify that predicted efficacy targets are achieved as soon as possible, ensuring that patients get the right drugs at the right dose as early as possible and before suffering side effects from poorly tailored treatment. This would be a dramatic improvement over current culture-based methods of regimen selection, where results return 2 months later, if at all. Our clinical site has previously enrolled ~800 participants with MDR-TB into cohort studies for longitudinal follow-up, monitored participants for side effects and treatment outcomes, and developed on-site workflows for phenotypic drug resistance, MIC testing, NGS from uncultured sputum, and plasma drug level testing of MDR-TB drugs. Each of these tools is available at our site, but they are only employed sporadically, are rarely used in the same patients, and have not been systematically analyzed for their relative contributions to patient outcomes. We will conduct a single-site observational cohort study of 210 adult participants with pulmonary smear-positive, rifampin-resistant TB to systematically evaluate the impact of a combination Baseline pRescription According to Direct from Sputum Sequencing and TArgeted drug Concentration Strategy (BRASS TACS) for personalized MDR-TB therapy as access to these tools expands. The specific aims of this proposal are to: 1) determine the proportion of patients with MDR-TB in Mumbai, India with resistance-associated mutations that would prevent treatment with moxifloxacin, linezolid, bedaquiline, clofazimine, or cycloserine using culture-free NGS; 2) identify the proportion of cohort participants with MDR-TB that achieve model-derived steady-state plasma levels meeting efficacy and toxicity targets; and 3) assess the time to final regimen, frequency of treatment-associated side effects, time to culture conversion, and final outcome of cohort participants who complete culture-free NGS and TDM. The results of this observational cohort will determine the combined benefit of these tools as they are deployed at a referral center with clinical and laboratory expertise in complex drug resistance. These data will describe the real-world outcomes of MDR-TB care using NGS and TDM for MDR-TB in a unique setting and will inform the future application of these tools to improved strategies for personalized MDR-TB treatment worldwide.

NIH Research Projects · FY 2025 · 2023-04

Project Summary/Abstract The dynamic writing and erasing of histone post-translational modifications on nucleosomes regulate eukaryotic gene expression by tuning chromatin organization and recruiting chromatin-binding proteins. The methylation of arginines can activate or repress transcription depending on the histone residue and its methylation state. Protein arginine methyltransferase 5 (PRMT5), along with its obligate binding partner Methylosome protein 50 (MEP50), is the primary complex for the symmetric dimethylation of arginine across all eukaryotes. In addition, PRMT5- MEP50 can either activate or repress the transcription of several genes, depending on which residue the enzyme modifies. PRMT5-MEP50 catalyzes methylation on four histone residues, namely histone H2A-Arg3 (H2AR3), H3-Arg2 (H3R2), H3-Arg8 (H3R8), and H4-Arg3 (H4R3). Due to PRMT5-MEP50’s diverse roles in transcription regulation, PRMT5 is overexpressed in several cancers as it regulates the transcription of several metastasis suppressor genes and epithelial-mesenchymal transition activating genes. Despite PRMT5-MEP50’s importance in gene expression, very little is known of how PRMT5-MEP50 methylates histone and/or nucleosome substrates. However, recent work has revealed that PRMT5’s specificity is regulated by (1) recognition of cytosolic H2A-H2B dimers to methylate H2AR3 and (2) being able to preferentially methylate histone H4 in the presence of substrate adaptor Coordinator of PRMT5 (COPR5). Despite these findings, molecular determinants towards this specificity are still unknown. Using a combination of biochemical and structural approaches, I will investigate the mechanism of histone specificity and activity by the PRMT5-MEP50 complex. In Aim 1, I will determine contributions towards PRMT5-MEP50’s recognition of H2A-H2B dimers by quantifying the activity and binding of PRMT5-MEP50 on various histone H2A-containing substrates. To provide molecular detail of this recognition, I will determine the structure of PRMT5-MEP50 bound to H2A-H2B dimers using cryo- electron microscopy (cryo-EM). While screening substrates of H2A methylation, I discovered that PRMT5- MEP50 activity is stimulated by ubiquitination of histone H2BK120 (H2BK120-Ub). I will probe in vivo relevance of this crosstalk by siRNA knockdowns. I will then reveal the mechanism of this activation by quantifying activity and binding of PRMT5-MEP50 in the presence of H2BK120-Ub and resolving the EM structure of PRMT5- MEP50 bound to H2A-H2BK120-Ub dimers. In Aim 2, I will elucidate the function of COPR5 and the PRMT5- MEP50-COPR5 complex. My preliminary data revealed that COPR5 does not bind to nucleosomes and cannot recruit PRMT5-MEP50 to the nucleosome, conflicting previous speculations of COPR5’s function. Therefore, I will identify COPR5’s preferred histone-containing substrate and quantify COPR5’s binding and contribution to the enzymatic activity of PRMT5-MEP50. Finally, I will solve the structure of PRMT5-MEP50-COPR5 bound to its histone substrate by cryo-EM. Together, this proposal will construct a molecular framework of PRMT5- MEP50’s substrate specificity to aid in structure-based drug design, by revealing substrate-specific interactions.

NIH Research Projects · FY 2026 · 2023-04

Each year, 20,000 children in the United States suffer a cardiac arrest, but only 17-50% survive. Survival is associated with high-quality chest compressions. Yet, healthcare providers adhere to the rate and depth guidelines recommended by the Pediatric Advanced Life Support (PALS) program for cardiopulmonary resuscitation (CPR) an abysmal 20-40% of the time. Rates of high-quality chest compressions improve to 64% with a quality CPR (qCPR) coach (a teammate giving real-time feedback on chest compression performance). While effective, qCPR coaching is resource and personnel intensive, limiting its use in prehospital or community settings where most children in cardiac arrest present for care. A novel strategy to provide real-time CPR feedback without additional personnel is the use of visual feedback in a medical provider's field of view through augmented reality (AR). An AR feedback system called AR- CPR has been shown to improve chest compression quality in real time, with promising usability and feasibility. Data from 34 subjects, demonstrate the feasibility of a head mounted display to offer instantaneous visual CPR feedback, and improved PALS adherence to chest compression rate and depth goals to 73%, from 17% without feedback. While these data are promising, significant development and evidence gaps remain. Therefore, the R21 aims of this proposal are to refine AR-CPR for accurate and precise rate and depth measurement (R21-Aim 1a), enhance usability (R21-Aim 1b), incorporate recoil feedback (R21-Aim 1c), and validate the accuracy and precision of all measurements (R21-Aim 2). The R33 aims of this proposal are to quantitatively evaluate AR-CPR in an international multicenter randomized simulation-based non-inferiority study as compared to qCPR coaching (R33-Aim 1) and qualitatively evaluate usability and user experience (R33Aim 2). It is hypothesized that these enhancements will improve the adherence to PALS guidelines and the rate of high-quality CPR performance, positioning AR-CPR as the most effective pediatric CPR feedback system available. The proposed research will take place as a multidisciplinary collaboration among investigators at The Johns Hopkins University School of Medicine, The Johns Hopkins University Applied Physics Laboratory, and the International Network for Simulation-based Pediatric Innovation, Research, and Education (INSPIRE). In accordance with the mission of The Agency for Healthcare Research and Quality (AHRQ), this proposal lays the groundwork to refine and improve AR-CPR by making this clinical lifesaving tool easier to use, accessible, and affordable and thus equitably supporting providers at the point of care to perform higher quality pediatric CPR in various clinical settings. This research will address two long term key goals: saving children's lives and addressing inequities in care delivery by making high quality CPR widely available in lower resource settings including in every hospital and ambulance, and with all automated external defibrillators in schools, airports, malls, and households.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Glaucoma is a chronic, progressive, incurable disease that affects over 2 million individuals in the United States alone and over 60 million worldwide. Sensitive detection of early glaucoma damage is not only essential for vision preservation, but also can facilitate new neuroprotective therapy developments. In this proposal, we focus on two novel imaging markers enabled by visible light optical coherence tomography (VIS-OCT), namely the reflectance spectral marker from retinal nerve fiber layer (RNFL) and macular oxygen consumption. We showed in our recent cross-sectional clinical study that peripapillary RNFL reflectance spectral marker and arteriovenous oxygen saturation difference in the macular region better separated early stage of glaucoma suspect/pre- perimetric glaucoma eyes from normal ones than circumpapillary RNFL and macular ganglion cell complex (GCC) thickness. These promising results lead to three specific goals in the project. First, we will develop a second- generation dual channel VIS-OCT device to improve the resolution, total imaging range, and image quality to achieve near shot-noise limit performance. Second, we propose to characterize the macular oxygen consumption by combining blood oxygen saturation and flow and correlated with glaucoma severity. Macular region contains >30% of total RGCs in retina and is involved in early events in glaucoma. The macular visual damage impacts the quality of life the most since it is at the center of our vision. For the first time, we will provide quantitative assessment of macular oxygen consumption in a spectrum of glaucoma severity and shed light in the pathological role of vascular function in the early-stage glaucoma. Finally, we propose a prospective study to correlate VIS-OCT imaging markers, including RNFL reflectance spectral markers and macular oxygen markers, with the glaucoma worsening. We will evaluate whether VIS-OCT markers can detect the early damages proceeding to vision functional loss at a later point. IMPACT ON PUBLIC HEALTH: The successful completion of this program will rigorously evaluate the clinical performances of new VIS-OCT markers for early glaucoma detection, which is highly impactful in clinical care and blindness prevention.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY The brain is built on billions of neural connections in a highly organized 3D hierarchy. At the same time, neural activity is highly dynamics that requires kilohertz imaging rate to capture action potentials and sub-threshold voltage signals, the fundamental bit for neural communication. While the recent advent of genetically encoded voltage indicators (GEVIs) makes it possible to optically record the neural membrane voltage, the technical challenges are profound in imaging millimeter-scale volumetric voltage imaging at kilohertz with cellular resolution. In this proposal, we aim to address the challenges by developing a one-photon mesoscopic (i.e. millimeter scale field of view, FOV) volumetric voltage imaging, using mesoscopic oblique plane microscopy (Meso-OPM). Our technique will image >1.8 mm2 FOV, >0.1 mm depth penetration at 1 KHz, capable of recording voltage signals across an entire nervous system of a Zebrafish larva. The bright and stable GEVIs Voltron with JF525 dye will be used in our proposed work. Meso-OPM is a variant of light sheet microscopy (LSM), with a single primary objective lens instead of two in conventional LSM. The simplified optical design allows 1) leveraging high photon efficiency in LSM; 2) integrating ultra-fast passive optical scanning to achieve >1 MHz frame rate; and 3) flexible optical designs for millimeter FOV and cellular resolution. In addition to the technical challenges for large-scale ultrafast 3D imaging, the effective data processing pipeline for massive data is also highly desirable. To this end, we propose a robust and efficient deep learning framework to perform self- supervised 4D denoising and neuron segmentation. The pipeline enable massive data processing at 10 volume per second for the downstream neuroscience studies. Finally, to demonstrate the utility of proposed techniques, we will image Zebrafish in response to optic flow by a drifting grating visual stimuli. We will identify neural circuitry responsible to the motion compensation to the optic flow (i.e. maintaining body position when presented drifting grating) from eyes all the way to spinal cord. Altogether, this proposal will greatly improve our capability of dissecting large-scale neural circuitry, and the sub-sequent modeling and creation of artificial neural circuits.

NIH Research Projects · FY 2025 · 2023-04

Project Summary: The overarching goal of this project is to provide Dr. Jackman with a rich training program that adequately prepares him to become an independent investigator focused on the development and implementation of digital healthcare interventions to address health disparities among minoritized populations. The United States has experienced record-breaking highs in sexually transmitted infection (STI) incidence and persistent disparities in STIs and human immunodeficiency virus (HIV) infections and sequelae among adolescent and young adult (AYA), low income, and Black/African-American populations. Serious adverse health consequences are attributable to STIs, including pelvic inflammatory disease, ectopic pregnancy, congenital syphilis, HIV, and infertility in an estimated 20,000 U.S. women annually. Patient centered clinical decision support (PC CDS) are personalized, patient-centric, patient-facing digital healthcare applications used to deliver patient care between health visits. This proposed program will leverage the electronic health record (EHR) patient portal in a novel PC CDS intervention to optimize sexual health decision making, partner communication, and uptake of clinical services among AYA aged 15 to 25 years in Baltimore, MD. Preliminary studies demonstrate the promise of EHR patient portal-based interventions to help youth overcome barriers such as, limited STI/HIV testing and treatment access, the stigma that often leads to non-disclosure, disclosure inaccuracies, and access to high quality and accurate STI/HIV-related information and resources. Recently, language included in the 21st Century Cures Act Final Rule, National HIV/AIDS Strategy, and STI National Strategic Plan bolsters patient portals as a critical tool for improving patient healthcare engagement. However, as reliance on patient portals and telehealth services have increased since the COVID-19 pandemic, so does the risk of perpetuating inequities in healthcare access among socioeconomically underserved youth. Yet, there is a considerable lack of interventions designed using a paradigm that amplifies the unique voices and experiences of minoritized AYA end-users to meet their healthcare engagement needs and preferences for STI/HIV prevention through the patient portal. This mentored research career development award is designed to provide the necessary training to excel in digital healthcare research focused on reducing health disparities and contribute to the public health informatics sciences. Research aims are to: (a) Describe longitudinal patterns and correlates of EHR patient portal use among urban AYA patients in a large academic health system; (b) Use a design thinking process to develop a PC CDS intervention to support the uptake of STI/HIV prevention services and behaviors among urban AYA patients via the EHR patient portal, and (c) Conduct a 3- month pilot study to determine feasibility and acceptability of the intervention. This study will provide crucial preliminary data to inform the design of an R01-funded randomized trial to determine the effectiveness of a novel PC CDS intervention to optimize patient portal engagement and reduce STI/HIV risk among urban AYA.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY Genetic diseases impact over 1 in 50 newborns worldwide and yet there are no approved therapies capable of correcting the underlying genetic defects. As a result, most patients continue to suffer throughout their life and require frequent interventions to ameliorate symptoms. I aim to develop in vivo genome editing therapeutics that correct the underlying disease mutation in relevant tissues by a single injection into the patient. Base editors can efficiently correct transition point mutations, the most common form of disease- causing genetic mutation, without undesired editing outcomes such as indels. With one dose and no subsequent enrichment, over 95% of cells in tissue culture can be edited, and editing in over 60% of non- dividing cells in targeted adult mammalian tissues has been demonstrated in early in vivo work. Prime editors can correct any genetic perturbation of up to at least ~50 nt in length (encompassing ~89% of human disease mutations). I will develop and assess both precision genome editing technologies (base and prime editors) using suitable in vivo delivery tools in mouse models to develop therapeutics for genetic disease. Dilated cardiomyopathy (DCM) is a frequent form of genetic heart disease, affecting an estimated 300,000 people in the United States, and can lead to heart failure. Common causes of DCM are haploinsufficiency of important genes in cardiomyocytes including TTN and LMNA. I will employ screens to identify new editing strategies to treat haploinsufficiencies by enhancing transcription of the healthy allele. I will characterize the mechanism of identified edits to understand the associated changes in transcription factor occupancy and chromatin states. Simultaneously, I will use fluorescent reporter mice to characterize the in vivo delivery of base editor and prime editor tools in order to find the best method for editing cardiomyocytes. This work will include characterizations of tissue- and cell-specific editing following delivery via adeno-associated virus, lipid nanoparticles, or polymer nanoparticles. I will then combine the identified therapeutic editing strategy with the best vehicle for delivery to cardiomyocytes to treat a mouse model of TTN haploinsufficiency. I will measure on- and off-target editing as well as any improvement in the contractility defect that defines this model. Base editors and prime editors can be readily reprogrammed to correct one or even multiple simultaneous mutations by altering the co-delivered guide RNA. Future work will expand this screening methodology to additional haploinsufficiency disorders, and applying identified delivery methods to new models of genetic disease. The ultimate goal of this work is to develop in vivo genome editing therapeutics that can be readily adapted to treat even rare or one-of-a-kind disease variants.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Brain activities involve neurons generating fast-propagating signals to encode and relay information within dynamic neural networks. Neuroscientists aspire to obtain access to such networks in unconstrained animal models (e.g., rodents) with high spatiotemporal resolution, which will shed light on the fundamental working mechanisms of the brain. Optical imaging, particularly multiphoton microscopy, has played a significant role in this endeavor. The past decade has seen impressive progresses, from head-restrained benchtop microscopy with virtual navigation to large FOV microscopy for neuron population imaging, three-photon microscopy for deep brain imaging, and two-photon (2P) miniscopy for in vivo imaging in freely-walking (but limited rotation) mice. Despite these exciting technological advances, tools for simultaneous, large-scale, and high-resolution imaging over multiple brain regions in freely-behaving rodents are still lacking. Successful development of such tools can accelerate the process of uncovering general principles of neural networks in a working brain under nearly natural conditions. The free-moving style for imaging would minimize the differences between experimentally controlled actions and natural spontaneous behaviors, thus allowing for precise examination of neural network functions. The capability of simultaneous imaging over two interconnected neural populations would provide a comprehensive and precise timeline of the neural circuit dynamics associated with various behaviors at both cellular and population levels. Our proposed research is motivated by the need for such imaging tools with the above-mentioned features. The main objective is to develop a 3D-scanning, ultrathin and light 2P fiberscope technology for enabling high- resolution, simultaneous imaging of dynamic neural activities over a large FOV at two brain regions in freely- moving rodents. To achieve our objective, we propose the following aims: (1) To develop a fast scanning 2P fiberscope of a large FOV (Ø500 um) using a cascaded magnification strategy while maintaining a compact probe size (Ø2.5 mm). The larger FOV will be achieved by using an innovative micro-optics design. In addition, a modular scanner head design will be implemented in the 2P fiberscope to improve the probe robustness for in vivo imaging at a high scanning frequency (e.g., ~2.8 kHz); (2) To develop a miniature (Ø2mm) tunable lens that can be integrated into our 2D scanning fiberscope for enabling depth (focus) scanning/selection over 150 um. Focus scanning allows for convenient selection of a proper layer or population of neurons. The tunable lens can create a curved refractive index profile when applied with a low-voltage (<10 V, safe) electrical drive. Compared with other tunable lenses, the tunable lens will be extremely compact and light, critical for imaging freely-moving rodents. A fiberscope integrated with a tunable lens will be developed and tested using phantoms, fluorescent tissue slides, and a mouse model in vivo. (3) To develop a dual-probe system, enabling simultaneous 2P imaging of two brain regions in freely- walking/rotating mice. The ultracompact size and lightweight of the fiberscope permit two fiberscopes to be mounted a mouse head, allowing for simultaneous imaging of two brain regions (cortex or deep brain). A novel, proactive, dual-probe optoelectrical commutator (dpOEC) will be developed for the first time to sense and compensate the torque built up in the fiberscopes, allowing the mouse to walk/rotate freely during imaging; (4) To assess the feasibility of the dual-probe 2P technology for exploring neural network dynamics in two different brain regions simultaneously during social decision making. Social behavior involves sensory, cognitive, and motor functions and thus depends on the interactions of many neurons, but until now no technology is available to record from a large population of neurons with subcellular resolution over multiple interconnected regions in freely-behaving mice. Here we choose to study the dynamic neural connectivity between the primary motor cortex (M1) and a critical sensory information routing node, periaqueductal gray (PAG). Both areas are critically involved in social behavior, but how these interconnected regions synergize to process information remains almost completely unknown. In addition to testing the performance of the 2P fiberscopy technology, this aim could also shed light on how social preference is encoded. As a control, we will monitor these regions during a locomotion (but nonsocial) activity (Rotarod running), for which the information on M1 that is independent of PAG is already available. In summary, successful completion of the proposed study will establish a new two-photon fiberscope imaging platform for the neuroscience community to enable simultaneous high-resolution imaging of neural network dynamics of different cell types over different brain regions in freely-behaving rodents. In addition, focus/depth scanning will be made possible. The fiberscope can be easily attached to and detached from the mouse head, permitting repeated use. Although beyond the scope of current proposal, the technology can also have many translational applications, including internal luminal organ imaging for diagnosis or guidance of intervention.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Critical to successful innovation in treating diabetes is the development of strategies for promoting insulin release and preventing pancreatic beta-cell destruction. Chronic demand for insulin production during insulin resistance and diabetes exacerbates cell dysfunction and this is compounded by ER and oxidative stress. This results in beta-cell death and loss of insulin production. Recent studies have highlighted defects in insulin processing, insulin granule maturation, and granule docking that are also linked to all major forms of diabetes; however conceptual gaps remain in understanding the causes of beta-cell failure and developing methods to reverse or prevent beta-cell dysfunction. Our preliminary studies establish Phosphatidylinositol transfer protein alpha (referred to as human PITPNA and mouse Pitpna), as a major regulator of insulin granule formation and secretion. PITPNA shuttles phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the trans-Golgi network (TGN) for phosphorylation by Phosphatidylinositol 4-kinase (PI4-K) conversion to phosphatidylinositol-4 phosphate (PtdIns-4-P), an abundant membrane phospholipid involved in insulin granule docking and exocytosis. Our preliminary data shows: 1) PITPNA expression is dramatically silenced in beta-cells of human T2D subjects, 2) reduction of PITPNA in human islets both lowered cellular PI4-P levels and insulin granule maturation and increased accumulation of proinsulin, and 3) conditional beta-cell specific deletion of Pitpna in mice (Ins-Cre; Pitpnaflox/flox) results in decreased insulin secretion and beta-cell mass, random-fed hyperglycemia, and increased expression of ER stress proteins in beta cells. Based on these data, we hypothesize that decreased PITPNA in beta-cells during T2D leads to lower PI4-P for distribution by the TGN as well as incorporation into insulin granules, thereby disrupting granule maturation, docking and secretion. We further hypothesize the reduced granule formation results in accumulation of proinsulin in the ER, leading to ER stress and ultimately beta-cell death. We propose that restoration of PITPNA in beta-cells of T2D individuals will reverse these aspects of cellular dysfunction. We expect these studies will demonstrate that promoting PITPNA function and PI4-P formation is a novel strategy for reversing beta-cell dysfunction in several subcellular compartments including the ER, mitochondria, and the TGN. These studies aim to highlight restoration of PI4-P between intracellular membranes as an innovative approach for increasing granule maturation and secretion as well as reversing beta-cell failure in major forms of diabetes.

NIH Research Projects · FY 2025 · 2023-04

PROJECT SUMMARY The cranial neural crest (NC) contributes to the formation of many craniofacial structures including the bones and cartilage of the face, tooth dentin and peripheral ganglia. Cell signaling regulates different aspects of cranial NC specification, epithelial-to-mesenchymal transition (EMT) and differentiation and disruptions in this developmental program results in many cranial NC-derived craniofacial birth defects including craniosynestosis, Treacher Collins and CHARGE syndromes, and cleft palate. BMP signaling plays a crucial role during the specification and differentiation of cranial NC, and more recently, BMP signaling was shown to control cranial NC EMT. A mechanistic understanding of the role of BMP signaling during cranial NC development is essential to develop novel preventative and therapeutic measures against craniofacial defects. This proposal will determine the molecular mechanism of BMP gradient formation in the chick gastrula, and how this gradient regulates the formation of cranial cell types including neural, cranial NC, placode and epidermal fates. These experiments will use in vivo and in silico approaches to test the hypothesis that extracellular BMP ligands are produced primarily by the cranial NC and are actively shuttled over long distances to signal most strongly in the nonneural ectoderm. Next, quantitative expression analysis and live imaging will be used to establish the timeline of BMP signaling during gastrulation and neurulation, and analysis of the resulting datasets will determine population- and single-cell-level responses to BMP signals. Differences in signal timing and strength will then be correlated with direct input into different target genes. Finally, the role of BMP target genes Id1/2/3/4 and Fibin during cranial NC EMT will be investigated using in vivo functional analyses. Together, the results of these aims will provide a comprehensive understanding of the regulation and roles of BMP signaling events during early cranial NC development. In addition to identifying targets for translational avenues to prevent craniofacial birth defects, the mentored phase of this proposal will provide Dr. Michael Piacentino with necessary training as he prepares to begin his independent career. Dr. Marianne Bronner's lab at California Institute of Technology, and his assembled advisory council, provide the necessary tools, expertise, and training environment to efficiently execute the proposed aims and establish Dr. Piacentino's independence. This training will be instrumental as Dr. Piacentino begins his independent research program and will provide the experience needed to make lasting impacts on the field of BMP signaling during craniofacial development.

NIH Research Projects · FY 2026 · 2023-04

ABSTRACT Childhood obesity is a high priority public health issue as it increases the risk of co-morbid diseases, including cardiovascular disease, fatty liver disease, and type 2 diabetes. An improved understanding of the factors that trigger the development of early life obesity is urgently needed. This is especially important among Hispanics, a minority group with high rates of obesity in early life. Beyond poor diet and a lack of physical activity, early life exposure to environmental chemicals, which are higher in underserved communities, independently contribute to childhood obesity. Human studies show that even at low levels of exposure during pregnancy, poly- and perfluoroalkyl substances (PFAS) are associated with rapid infant weight gain and greater risk for childhood obesity. Postnatally, breastfeeding is a primary source of inadvertent PFAS transmission to infants, potentially offsetting some benefits of extended breastfeeding. Recent findings suggest that the developing gut microbiome is exposed to breast milk PFAS, which may alter gut bacteria and fecal metabolites that contribute to obesity. Despite this, human studies have largely focused on prenatal PFAS exposure, and no prior studies have examined the effects of breast milk PFAS on rapid infant growth and the gut microbiome during infancy, a critical period in which interventions have the potential to prevent the development of childhood obesity. Our overarching hypothesis is that higher concentrations of breast milk PFAS contribute to more rapid infant growth and childhood obesity risk, and that these effects are explained by alterations in the composition and function of the infant gut microbiome. This hypothesis is based on results from our preliminary data, which demonstrate that infant gut bacteria are associated with infant weight and breast milk PFAS at 6-months of age. Our multidisciplinary team of investigators propose to test this hypothesis in a cohort of 208 Hispanic mother-child pairs with assessments of child growth at 1, 6, 12, 18, 24, and 36-months as well as at 6yr of age. This study will measure breast milk PFAS concentrations and characterize the infant gut microbiome and fecal metabolome using archived breast milk and stool samples at 1- and 6-months to advance our mechanistic understanding of the obesogenic effects of PFAS exposure while accounting for prenatal PFAS exposure using newborn dried blood spots. Our aims are to determine the extent to which early life exposure to breast milk PFAS are associated with: 1) child weight from 1-month to 6 years (Aim 1A) and the risk of rapid growth and childhood obesity (Aim 1B) as well as 2) changes in gut microbial profiles and fecal metabolites (Aim 2). Our ultimate goal (Aim 3) is to integrate breast milk PFAS exposure, gut microbiome, and fecal metabolomics profiles to identify subgroups of children that are at increased risk for rapid growth and obesity. This study offers a unique opportunity to advance our understanding of breast milk PFAS and may identify preventive measures that could be used to offset obesity-risk, including screening for breast milk PFAS and the use of probiotics to promote growth of beneficial gut bacteria in early life.