Johns Hopkins University

NIH Research Projects · FY 2025 · 2022-09

Project Abstract: Recent research has highlighted the importance of human associated microbiota in many diseases and health conditions. Nowadays marker-gene amplicon and shotgun metagenomics sequencing (jointly, MGS) have been routinely used in epidemiological and clinical studies to investigate the health impact of the microbiome commu- nity. In the public domain, many researchers now deposit MGS data together with other data for other researchers to investigate. Despite being increasingly available, MGS data analysis remains difficult. In addition to the classic statistical challenges inherent to MGS data such as the compositionality, the sparsity, the over dispersion and the phylogenetic relationship between taxa, large scale MGS studies feature additional complications including the experimental bias and hidden artifacts (batch effects), which will invalidate downstream analysis if not accounted for properly. Current analytic approaches largely ignore or insufficiently handle these difficulties. This proposal aims to develop powerful and robust statistical methods for reproducible microbiome discoveries that adjust for unknown batch effects and are resistant to sequencing biases. In aim 1, we will develop a novel approach to search for unmeasured artifacts through a novel surrogate variable analysis and multiple quantile thresholding. Our approach advances the existing surrogate variable analysis approach to specifically address the characteristics of MGS data including the differences in variabilities, the sparsity and the zero inflation. In aims 2 & 3, we develop bias resistant modeling for assessing microbiome-phenotype association and community level analysis. We will also develop, distribute and support user-friendly software for the proposed methods to benefit the entire research community. The proposed methods will be evaluated against extensive simulations and analysis of real microbiome data including data from our motivating studies as in VAPing Observational Research Study (VAPORS) and the New Hampshire birth cohort study. Successful completion of this proposal will fill the gap between the increasing research interest in microbiome and the lack of robust and bias-resistant tools, and facilitate our in-depth understanding of human microbiome in health and disease.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY: Despite advances in treatment and prevention over the past decades, human immunodeficiency virus (HIV) imposes substantial burdens in the United States (US). US HIV infections are increasing concentrated in Southern states with a disproportionate number of rural infections, particularly among racial, ethnic, and sexual minorities. Rural epidemics in these states are linked to high-intensity urban epidemics, and driven by racial/ethnic disparities, poverty, inadequate insurance, limited access to healthcare, and housing insecurity. Additionally, the opioid epidemic disproportionately affects rural communities, increases HIV transmission, and hinders HIV control efforts. The Ending the HIV Epidemic (EHE) Initiative seeks to reduce HIV incidence by 90% over a decade. Ending the US HIV epidemic will require interventions tailored to local-level epidemic dynamics that address underlying drivers of transmission. Mathematical models of HIV transmission are powerful tool to forecast epidemics, and can provide evidence-based guidance on the optimal way to prioritize limited public health resources, but these models have focused primarily on urban epidemics. We have previously developed the Johns Hopkins Epidemiologic and Economic Model (JHEEM), a platform for modelling local HIV epidemics. Our objective is to generate projections of local HIV epidemics in states with a high rural burden of HIV to inform policy decisions by local health departments. We will partner health departments in three states where we have established relationships (Alabama, Louisiana, and Mississippi) to develop a suite of transmission models, based on JHEEM, that rigorously leverage local surveillance data to make projections of the HIV epidemic in each state. In Aim 1, we will develop an integrated, statewide modeling approach that links rural and urban regions using mobility data. We will use the models to identify which combinations of HIV testing, pre-exposure prophylaxis (PrEP), and viral suppression – targeted to which demographic subgroups and geographic regions – will yield the greatest reductions in incidence and disparities. In Aim 2, we will incorporate three social determinants of health: insurance, access to healthcare, and housing instability, and the racial and ethnic disparities in their distribution across the population. We will project the impact on HIV incidence and disparities of strategies that include both traditional HIV interventions and increase insurance, access to care, or stable housing. In Aim 3, we will expand JHEEM to include the opioid epidemic, and evaluate the potential reductions in HIV incidence achievable by strategies that combine harm reduction for opioid use disorder with HIV control interventions. These aims will yield a comprehensive modeling framework that links traditional HIV interventions with underlying drivers of the epidemic in rural America. Our results will provide data-driven projections, tailored to the specific needs of states, to inform effective deployment of public health resources in ending the HIV epidemic.

NIH Research Projects · FY 2025 · 2022-09

Dr. Page is a Professor of Medicine and infectious disease specialist engaged in clinical care, and public health research with a focus on expanding access to care and quality of care through community-engaged program building and evaluation. Over the last 15 years, her research has focused on: 1) Elucidating the impact of socioeconomic stressors on health; 2) Implementing and evaluating tailored interventions to improve access and quality of care; and 3) Bridging the gap between scientific advances and real-world implementation to reach communities at highest risk of disease. She has established a community coalition that has developed low-cost interventions to improve health outcomes. The objectives of this application are to support a program for mentoring junior investigators interested in community health and to foster innovative epidemiology and implementation science research through interdisciplinary and community-engaged approaches. The research strategy proposed in this award capitalizes on existing multi-stakeholder community-academic-public health partnerships and multiple independently funded research studies. This Mid-Career award explores the multi-level barriers and facilitators of engagement in healthcare and research through peers and technology (hybrid methods) that leverage social networks, mobile health (mHealth) and trusted community health workers. Informed by the Information-Motivation-Behavioral Skills model (IMB) and Social Network Theory, Aim 1 will determine the efficiency and acceptability of hybrid methods to promote healthcare engagement. In Aim 2, the study will determine the efficiency and acceptability of using hybrid methods for recruitment, data collection, and research engagement by comparing traditional convenience sampling approaches to sampling using technology-based methods. Using the Consolidated Framework for Implementation Research (CFIR) and Research Effectiveness-Adoption Implementation Maintenance (RE-AIM) framework, semi-structured interviews will be conducted with key stakeholders to assess and inform implementation determinants and outcomes of hybrid methods to inform future broad scale implementation (Aim 3). This proposal creates a platform for trainees to learn and apply skills in community-based research, implementation science, mHealth, network analysis, epidemiology, and health disparities. This work also strengthens the research pipeline by offering community-engaged training opportunities for early-stage investigators, especially those with a commitment to improving health outcomes through implementation science.

NIH Research Projects · FY 2024 · 2022-09

Transplantation of therapeutic cells holds great potential to cure or provide relief to various ailments. In the clinic, cell grafts often perish or cease to function within a short period post-transplantation. An emerging strategy is the use of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) as an adjuvant to cell therapy. However, there remain challenges for effective EV delivery to and retention at the target sites, and the inability to elucidate the biodistribution and pharmacokinetic profiles of EVs using a clinically relevant tool. Moreover, a few new studies indicated that multiple deliveries of EVs are essential for therapeutic outcomes, which are hard to accomplish using the current clinical administration routes. We propose to develop theranostic, injectable microspheroid EV-delivery systems (EVDS) for local, targeted, and sustained-delivery of EVs while enabling EV tracking in vivo with magnetic particle imaging (MPI). The design of our EVDS is modular where each main component of the system can be independently modified to suit different purposes. Our interest lies in pancreatic islet transplantation to treat type 1 and advanced type 2 diabetes. Two designs of EVDS – LipoCap and MICap – are proposed. LipoCaps (500 μm) can be infused into the liver via the portal vein (a clinically tested islet transplantation protocol) for co-implantation with islets, or for follow-up doses post-transplantation. MICaps (900 µm) are appropriate for implant site with larger volumes, such as the intraperitoneal cavity. Both designs aim to obtain a local, sustained delivery of EVs to islets. LipoCaps and MICaps will be composed of ultrapurified alginate, an FDA-approved excipient of food, cosmetic and pharmaceutical products. In order to minimize non- specific uptake by nearby fat and tissues after the release from alginate matrix, EVs will be loaded inside islet- targeting liposomes prior to alginate encapsulation. This will be achieved by conjugating the liposomes to exendin-4, a ligand of glucagon-like peptide-1 receptors which are abundantly expressed on the surface of islet beta cells. In addition, EVs will be labeled with clinical-grade ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) to facilitate imaging by MPI. MPI is an emerging modality that provides “hot spot” visualization as well as EV quantification, much alike to PET and SPECT without the use of radioactive agents. Unlike 1H MRI, background artifacts from blood pools and motion effects are not an issue with MPI. MPI is reported to be more sensitive than 19F MRI and CT, and therefore may reduce the quantity of labels introduced into the patients. MPI may inform physicians on the temporal and spatial behavior of EVs which, in turns, may afford EV therapy to be customized. To systemically investigate the feasibility of the project, three specific aims are proposed: 1) To synthesize and characterize LipoCaps and MICaps carrying MSC-EVs, and to test their effects on human islets’ survival and function in vitro; 2) To develop a USPIO-labeling method that will preserve EV properties while maximizing MPI detection sensitivity; 3) To test if the proposed EVDS can improve islet survival and function in immunodeficient NU/J mice while being tracked by MPI.

NIH Research Projects · FY 2025 · 2022-09

Modified Project Summary/Abstract Section While preventive care guidance recommends primary care clinicians deliver reproductive health (RH) care to male adolescents, males’ RH care receipt is poor. Clinic-based interventions can be valuable RH promotion tools for adolescents. Yet, they have mainly focused on RH care for girls or single topics rather than the recommended RH care package for males. Computer based approaches make it easier to consistently provide evidence-based RH education, in multiple languages, tailored to individual needs. Using such approaches before the clinic visit can also make it easier for clinicians’ work flow, such as time constraints and discomfort in discussing RH. Yet, we are not aware of any computer-based strategies for use in the clinical setting to promote recommended RH care for male adolescents. Neglecting to provide males with evidence-based RH care fails to meet their own needs, and compromises their partners’ health. Health-E You/Salud iTu is a pre-visit, individually tailored, interactive, web-based mobile intervention shown to improve hormonal contraceptive knowledge, self-efficacy, and use six months later. It also primes and extends clinicians’ ability to deliver individually tailored care to patients. The proposed study will adapt the current Health-E You for male adolescents to assess their RH needs; provide interactive, individually tailored, evidence-based RH information; support and prepare males’ RH decision-making and visit priorities; and support clinicians’ ability to individually tailor recommended care. We will then evaluate its acceptability, usability, satisfaction, and efficacy on RH care receipt and method use among male adolescents presenting for care at primary care settings. In this R01 proposal, we propose to (1) adapt Health- E You for use with male adolescents using an iterative Youth-Centered Health Design Process (YCHD) with males and input from an advisory board of male adolescents and clinicians; (2) ensure perceived acceptability, usability, and satisfaction of the final Health-E You app among male adolescent patients and clinicians using a YCHD approach; and (3) test its efficacy to improve sexually active male adolescent patients’ knowledge, self-efficacy, and behaviors related to RH care after the visit and method use 2 months later. The current proposal would be the first to examine the acceptability, usability, satisfaction, and efficacy of a pre-visit, computer-based intervention to engage male adolescent patients in RH care and method use, where currently no such strategy exists.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Shoulder pain is extremely common after stroke and occurs in 30-70% of patients. Chronic post stroke shoulder pain (PSSP) contributes to depression, interferes with motor recovery, and decreases quality of life. Although PSSP is thought to be caused by damage to the myofascial tissues around the shoulder joint, the pathophysiology of myofascial dysfunction and pain in PSSP has not been elucidated, leading to missed opportunities for early diagnosis and variable success with pain management. The accumulation of hyaluronic acid (HA) in muscle and its fascia can cause myofascial dysfunction. HA is a glycosaminoglycan (GAG) and a chief constituent of the extracellular matrix of muscle. In physiologic quantities, it functions as a lubricant and a viscoelastic shock absorber, enabling force transmission during muscle contraction and stretch. Reduced joint mobility and spasticity can result in focal accumulation and alteration of HA in muscle, leading to the development of taut bands, dysfunctional gliding of deep fascia and muscle layers, reduced range of motion (ROM), and pain. Muscle HA concentrations can be imaged using T1rho (T1ρ) MRI, and myofascial dysfunction can be assessed using echo texture analysis and shear strain mapping on quantitative ultrasound (US), which may serve as useful biomarkers to elucidate the pathophysiology of myofascial dysfunction in PSSP. Hence, in the R61 phase we will: (1) Quantify the extent of GAG/HA accumulation using T1ρ MRI in the paretic versus non-paretic shoulder rotator muscles, and correlate the T1ρ MRI measurements with US echo texture measurements to develop a clinic-friendly tool to infer the extent of HA accumulation; and (2) Distinguish between latent versus active PSSP using US shear strain mapping of the same muscles on the paretic side compared with the non-paretic side during passive shoulder external rotation (ER), which is strongly associated with PSSP. To proceed to the R33 phase, we will demonstrate a statistically significant difference in (1) GAG/HA accumulation using T1ρ MRI, and (2) shear strain mapping for latent and active PSSP using quantitative US. In the R33 phase, we will use the imaging metrics identified in the R61 phase to monitor treatment response in a clinical trial of intramuscular (IM) hyaluronidase injections to increase pain-free passive shoulder ER-ROM. We will administer human recombinant hyaluronidase or normal saline injections in the dysfunctional shoulder girdle muscles, and measure pain-free shoulder ER-ROM (primary outcome) (1) pre-injection, (2) 1–2 weeks post-injection (primary endpoint) and (3) 6-8 weeks post-injection. We expect that local hyaluronidase injections will breakdown the accumulated HA leading to increased pain-free shoulder ER, and that the improvement will correlate with quantitative MRI and US imaging metrics. At its conclusion, this proposal will develop quantitative imaging biomarkers of myofascial dysfunction to monitor response to treatment and aligns with the NIH HEAL Initiative to bolster research to enhance pain management.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Type 1 diabetes (T1D) is a chronic disease that requires intensive daily management to maintain adequate glycemic control and prevent diabetes-related complications. Less than 20% of individuals meet the American Diabetes Association goal hemoglobin A1c level for adequate glycemic control. Use of diabetes technologies, and specifically continuous glucose monitors (CGMs), are the standard of care, and improve glycemic control, health related quality of life and treatment satisfaction. Despite these benefits, CGMs are underutilized, particularly in minorities and individuals from underserved communities. Increasing uptake and continued use of CGM can help improve glycemic control and mitigate disparities in diabetes-related outcomes for underserved individuals. There are many barriers to CGM use at the patient, provider and systems levels, yet there are no proven interventions to address these barriers. In a pilot study at our institution, we demonstrated that placement of trial CGM with standardized education at the point of care increases CGM uptake, but overcoming the knowledge barrier is insufficient to ensure personal and sustained use of CGM. The objective of this project is to build upon our pilot study to implement a novel intervention in the real-world clinical setting that is feasible, sustainable and generalizable to increase CGM uptake across the lifespan - including pediatric, emerging adults, and adults with T1D. We hypothesize that implementing the support of a diabetes navigator with trial CGM placement at the point of care will increase CGM uptake and sustained use, leading to improvements in glycemic control that will mitigate disparities in diabetes related outcomes for underserved individuals with T1D. In this proposal, we will first (Aim 1) refine the role and toolkit of the diabetes navigator based on stakeholder feedback on solutions to address barriers to CGM uptake and sustained use. Building on the experience from our pilot study, the formative phase of Aim 1, and leveraging the collective expertise of our multidisciplinary study team, we will conduct a randomized controlled trial (Aim 2) of 136 individuals with T1D, including 60 children and adolescents and 76 adults, not currently using CGM. Participants will be randomized to (Arm 1) standard of care trial CGM placement (n=68), or (Arm 2) the intervention arm with trial CGM placement with the support from a diabetes navigator (n=68). We hypothesize that the diabetes navigator arm will have higher CGM uptake and sustained use, with (Aim 3) greater improvements in glycemic control and patient-related outcomes, compared to the standard of care arm. If the diabetes navigator is successful in increasing CGM uptake and sustained use, and is demonstrated to be a feasible, acceptable and sustainable intervention that can be applied to the larger diabetes community, it has the potential to improve diabetes care and mitigate disparities in diabetes related outcomes for underserved communities with T1D.

NIH Research Projects · FY 2025 · 2022-09

Abstract: Increasing evidence supports the role of metal exposures as a ubiquitous source of neurological diseases, including Alzheimer’s Disease (AD) and related dementia, in multiple populations worldwide. In real life, one is more likely to be exposed to metal/metalloid mixtures than individual metals. The US EPA has designated the ubiquitous combination of Pb, As, Cd, and Cr(VI) (PACC) as the top interaction profile of concern for exposure. However, there is still a critical knowledge gap on the adverse outcome pathways (AOPs) due to chronic exposure to mixtures of metals/metalloids, which hampers risk assessment of neurodegenerative diseases. The goal is to elucidate the mechanisms by which metal mixtures act in concert to elicit neurodegenerative AD effects to support the cumulative neurodegenerative disease risk assessment. Our preliminary data from in-vivo mouse experiments indicate that, unlike single metal exposures, chronic PACC mixture exposure during adulthood has sex-specific negative effects on cognition, memory, and anxiety. This correlated with increased serum neuronal decay biomarker (NFL-a), neuroinflammation, and imbalanced redox homeostasis due to altered Nrf2 signaling. In human brain organoids, the PACC mixture increased oxidative stress, while it reduced the expression of the pre-synaptic marker, Syn1, and the neuroprogenitor marker, Nestin, at different stages of organoid development. We hypothesize that interaction profile PACC metal mixtures relative to single metals at or below regulatory limit impairs brain development as well as accelerates cognitive decline, neurodegeneration, and eventually AD, due to epigenetic changes in interconnected inflammatory and oxidative stress pathways. We will test this using the following aims: SA 1: To determine the biological mechanisms by which exposure to PACC metal mixtures during adulthood cause AD-related neurocognitive decline. The cumulative risk of sex- and dose-specific neurotoxicity and neurodegeneration caused by adulthood exposures to individual metals vs. PACC mixtures will be determined using mouse models. The causality of oxidative stress and the Nrf2/KEAP1 pathway will be tested using Nrf2fl/fl or Keap1fl/fl knock-out mice. SA 2: To investigate the adverse neurological effects of perinatal exposure to PACC metal mixture at environmentally relevant doses. We will determine the neurological effects of PACC metal mixture in perinatally exposed adult mice and perform a cumulative risk assessment. SA 3: Determine potential gene x environment (G×E) interactions involved in PACC metal mixture-induced AD-related neurodegeneration. Using our CRISPR/Cas9 modified human organoids in which the AD risk gene APOEe4 is knocked out, we will study metal combinations at different stages of brain maturity, and investigate oxidative stress, transcriptomic and metabolic changes, and associated neurodegeneration. Impact: This study will provide insights into the long-term impact and causal mechanisms of metal mixture- induced neurotoxicity and help tailor risk management decisions to protect populations from metal-induced AD.

NIH Research Projects · FY 2026 · 2022-09

Recent animal studies have provided new evidence that the cerebellum may have a stronger link to the reward system of the brain than was previously recognized. Direct projections from cerebellar deep nuclei (DN) to the ventral tegmental area (VTA) have been identified, and stimulation of these cerebellar afferents to the VTA was found to be rewarding. Such findings raise the possibility that cerebellar dysfunction could contribute substantially to addiction via a cerebellar influence over VTA. Consistent with animal findings, we have found in human fMRI preliminary data strong cerebellar and VTA activation in response to alcohol cues relative to non- alcohol stimuli in patients with alcohol use disorder (AUD) compared to controls, and close coupling observed between DN and VTA activation. Studying AUD and control participants, this project will address three important questions. The first is: What is the nature of cerebellar input to the VTA, and how is it perturbed in AUD? A number of investigations have suggested that when a stimulus is presented, the cerebellum generates a prediction of events that will follow based on prior associative learning, and then compares predicted and actual outcomes to generate a prediction error. We hypothesize that these functions are disrupted in AUD. Our preliminary data show that when an expected stimulus does not occur, a strong prediction error signal in the form of increased functional connectivity (FC) between cerebellum and its projection target is observed, and we found an analogous increase in DN-VTA FC, that was abnormal in AUD patients, when alcohol pictures were presented. In Aim 1, using fMRI and a monetary incentive task, we will investigate if DN-VTA FC reflects reward prediction and/or positive or negative reward prediction error. The second question is: Is the amount of activation in brain reward centers that is elicited by alcohol stimuli related to the amount of dysfunction in the cerebellum? In Aim 2 we will investigate 2 measures of cerebellar integrity to determine their relationship with the magnitude of alcohol cue related activation in cerebellar, VTA, and other reward structures, and with DN- VTA FC: (1) The timing of the undershoot of the cerebellar hemodynamic response function (HRF), which has been found to be correlated with number of lifetime drinks; and (2) classical eyeblink conditioning, for which the cerebellum is necessary. The third question is: Can abnormal cerebellar activation and FC, as well as alcohol craving, be reduced by non-invasive cerebellar stimulation? In Aim 3, Using fMRI combined with cerebellar transcranial direct current stimulation (tDCS) during a cue reactivity task, we hypothesize that in AUD participants cerebellar and VTA activation will be reduced, DN-VTA FC will be normalized, and alcohol craving will be reduced. We will examine, using both resting state fMRI and psychophysiological interaction analysis, the effects of tDCS on FC among important structures of the reward system as well as on DN-VTA FC. These investigations will lead to a better understanding of the involvement of the cerebellum in AUD, as well as the therapeutic potential of cerebellar modulation.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Undernourished women in low-and-middle income countries (LMICs) experience greater fetal growth restriction in pregnancy and poorer birth outcomes, including preterm birth and small-for-gestational age (SGA). Infants born preterm and/or SGA are more vulnerable to stunting and inadequate neurocognitive development, with lasting effects through adulthood. Nutritional interventions targeted in pregnancy and lactation have the potential to break the intergenerational cycle of malnutrition. Recently, an expert consensus developed specifications for a fortified BEP “ready-to-use” food supplement for pregnant and lactating women in LMICs, which requires testing in undernourished settings for efficacy in improving birth and growth outcomes. Currently, there is no recommendation for supplementation for women who are breastfeeding, despite high rates of growth faltering in early life in LMICs, where maternal undernutrition is high and exclusive breastfeeding is recommended. Our research aims to 1) Evaluate the effect of daily BEP++ in pregnancy and/or lactation on infant growth outcomes in the first 6 months of life; 2) Evaluate the effect of daily BEP++ in pregnancy and/or lactation on maternal nutritional status and explore potential mediation pathways on infant growth outcomes; and 3) In a biospecimen sub-study, explore relationships between maternal and infant nutrient status, inflammation, human milk composition, and gut microbiome by supplementation in pregnancy and/or lactation vs. not. We propose a community-based, randomized controlled trial of daily BEP++ supplementation during lactation in rural Nepal (proposed addition). Our study population has high rates of maternal undernutrition, adverse birth outcomes, and poor infant growth, and is representative of rural settings in South Asia. To conduct this trial, we will leverage the infrastructure of our community-based field site and a randomized trial of a BEP++ supplement in pregnant women scheduled to begin in 2022 (parent trial). The parent trial will recruit pregnant women in pregnancy, randomize them to supplement in pregnancy or no supplement, and follow-up until birth to assess SGA incidence. We propose to 1) add a postpartum supplementation trial following supplementation in pregnancy, using a 2x2 factorial design, to evaluate the BEP++ supplement during lactation and measure infant growth outcomes at 6 months of life, 2) double the overall sample size of pregnant women enrolled to allow for detection of meaningful differences between postpartum supplementation, and 3) nest a biospecimen sub-study within the trial to explore aims on the causal relationships between maternal and infant nutritional status and infant growth. A sample of 2,000 pregnant women will yield 1,600 live births and 1,440 infants followed until 6 months (accounting for pregnancy loss and loss to follow-up). This will allow us to detect a difference for our primary outcome, mean length-for- age Z score at 6 months, between supplement in lactation vs. no supplement, of 0.16 Z scores.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Infectious disease is a major threat to human health worldwide. The emergence of antibiotic resistance pathogens necessitates the development of new drugs to treat infections. A fundamental challenge in developing antibiotics is that many pathogens replicate inside host cells rendering them inaccessible to antimicrobial agents. The critical processes that govern pathogen growth within host cells represent promising new targets for therapeutic intervention. Most bacterial pathogens that grow inside host cells do so in a specialized compartment called the replication vacuole. Maintaining the integrity of the replication vacuole is critical to bacterial survival and growth as it provides protection against host surveillance systems that detect and eliminate pathogens. Disrupting this process would thus limit bacterial growth and enabling pathogen killing by the host immune system. Recently, we discovered an unprecedented link between host cell peroxisomes and the ability of the bacterial pathogen Legionella pneumophila to sustain its replication vacuole. The impaired growth of Legionella in the absence of peroxisomes demonstrates a critical role for peroxisomes in supporting Legionella infection. Peroxisomes play central roles in numerous cellular processes. Importantly, peroxisomes are most widely recognized for their critical functions in the production and breakdown of lipids, major constituents of pathogen replication vacuoles and sources of nutrients for growing bacteria. This suggests that Legionella may exploit peroxisomes to gain access to these essential molecules. In addition to Legionella, many pathogens that grow within host cells, use common strategies to establish an infection, in particular the formation and maintenance of a replication vacuole. Thus peroxisomes are likely to be important for other infectious bacteria. The proposed research will define how peroxisomes enable Legionella to cause disease while simultaneously avoiding detection and elimination by the body’s natural defenses, which is paramount to the development of new antibiotics.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The complexity of mammalian embryogenesis makes it challenging to determine the effect of genetic perturbations on development. The long-term goal is to better understand how genetic and environmental factors alter mammalian devel- opment to affect adult phenotypes or cause diseases. Toward achieving this long-term goal, the overall objective of this application is to develop a platform for high-throughput retrospective lineage reconstruction to quantitatively map devel- opmental alterations in a mouse model of autism. This platform will be based on developmental barcoding where random mutations accumulate in synthetic loci during embryogenesis. Each mutation is inherited by the descendants of the cell in which it occurs; each descendant can add new mutations to the ones it inherited. This process marks each cell with a set of mutations—a barcode—that can be used to resolve its lineage. The central hypothesis is that high-resolution lineage barcodes that are sequenced spatially in single cells can be used to retrospectively map proliferation and differentiation dynamics of neural progenitors to identify the differences between wildtype and mutant mouse models. The rationale for this research is that many genetic risk factors that are associated with birth defects remain mechanistically inexplicable based on cellular and molecular analyses of terminally differentiated tissues; this platform would enable retrospective mapping of these genetic effects after development to determine which progenitors they affect, when they affect those progenitors, and how they affect the behavior of those progenitors during development. The central hypothesis will be tested by pursuing three specific aims: 1) Establish high-resolution lineage recording in combinatorial and cumulative bar- codes embedded in each cell’s genome. This Aim will combine mutagenesis from double-strand breaks, which predomi- nantly lead to indels, with orthogonally induced point mutations to establish ultrahigh resolution lineage recording throughout mouse gestation. 2) Establish in situ single-cell barcode and identity readout directly from mouse tissues. This Aim will engineer barcoding loci to facilitate their amplification and sequencing in tissue sections together with molecular markers of cell state. Combining cell state and lineage barcodes will reveal proliferation and differentiation dynamics of their progenitors. 3) Determine the effects of Chd8 haploinsufficiency on the development of mouse brain using retro- spective lineage reconstruction. Chd8 haploinsufficiency causes autism but how it alters neurogenesis remains unclear. This aim will quantify the effects of Chd8 haploinsufficiency on proliferation and differentiation parameters of brain pro- genitors during mouse neurogenesis. The research proposed here is innovative because it establishes new strategies for high-resolution genomic barcoding of lineages and high-throughput spatial sequencing of these barcodes in tissue sec- tions. It further uses new theoretical concepts to convert terminal cells’ lineage barcodes and molecular identity infor- mation to quantitative insights about their progenitors. Additionally, it carries out in utero analysis of how Chd8 haploin- sufficiency alters progenitor fields that create the brain. This research is significant because it will enable determining how genetic perturbations alter mammalian embryogenesis to cause developmental anomalies such as autism.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Bipolar disorder (BP) is a severe multifactorial neuropsychiatric disorder that imposes a significant burden on public health. The most recent large-scale genetic study of BP identified 64 associated genetic loci, providing initial insights in BP pathogenesis. Yet, genetic discovery in BP lags behind other key psychiatric disorders. The reported genetic loci only capture a small proportion of the total BP genetic liability, with many more variants across the common and rare allele frequency spectrum remaining to be discovered. In addition, the previous studied samples were of European ancestry, leaving population specific BP variants uncovered and uncertainty in how the BP genetic findings generalize to other populations, exacerbating healthcare disparities, and these studies rarely employed “deep” phenotyping or assessed relevant environmental risk factors. This proposal brings together an international collaboration of leading investigators from the U.S., Taiwan, South Korea, Singapore, India, and Pakistan to form the Asian Bipolar Genetics Network (A-BIG-NET) and carry out a large- scale genetic study of BP in East and South Asia. A-BIG-NET will generate a BP genetic resource of 27,500 cases and 16,000 controls with rich phenotypic information, measures of key environmental stressors and genetic data from 4x low-pass whole genome sequencing (4xWGS). This will complement a schizophrenia genetics resource of 22,778 cases and 35,362 controls of Asian ancestry previously assembled by leaders of this network that will be available for cross-disorder comparisons. Studying BP genetics in Asia is important to the world and the U.S., as Asia constitutes 57% of the world population, and Asian American comprises 6.6% of the U.S. population (21.4 million). The five countries in A-BIG-NET cover 35% of all Asian populations. The specific aims of the proposal are to: 1) recruit and deeply phenotype 17,500 BP cases, with a focus on BP-I to maximize homogeneity, and 14,000 controls from four Asian countries; 2) carry out 4xWGS on all recruited samples plus 10,000 BP-I cases and 2,000 controls collected by a previous study using similar procedures in Pakistan; and 3) carry out a range of analyses to discover new genetic associations with BP-I across the allelic spectrum in East and South Asian populations, examine the comparative genetic architecture of BP-I across major world populations and with other major neuropsychiatric disorders, and perform a novel statistical fine- mapping analysis that leverages the multi-ancestry genomic diversity and pleiotropy across psychiatric disorders to identify putative causal variants. Aim 3 will also explore the genetic “validity” of various BP-I subtypes and fit models with joint genetic and environmental risk factors. This proposal will dramatically increase the worldwide diversity of genetics data on BP, an important step to accelerate gene discovery in this disorder and advance global mental health equity.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Temporal bone research is essential for refining our understanding of the mechanisms of otologic disease; however, the difficulty in accessing this tissue, the typically long processing times, and the expense have contributed to a dwindling number of labs engaged in this work. In the 1920s, the first human temporal bone laboratory in the U.S. was established at Johns Hopkins. The collection contains approximately ~1400 processed specimens from donors with a range of otologic diseases that were obtained primarily in the 1920s through 1970s, but only a handful of studies have made use of this collection in the past 20 years. We propose to make the collection more accessible to the scientific community and to re-establish the infrastructure for harvesting, imaging, and processing new specimens with the intention of recruiting new donors from population-based studies of aging in which participants have already consented to autopsies and brain donations being performed at Johns Hopkins, as well as other clinical otological studies ongoing in the Baltimore Metro Region. We will partner with faculty specializing in biological visualization, bilingual scientific communication, and mentoring diverse scientists to develop inclusive outreach tools. We will also partner with Baltimore-based transcriptomics resources to establish protocols for performing spatial transcriptomics analysis in nonhuman primate and human temporal bones. We will approach these objectives with three Specific Aims: 1) Catalog, prioritize, and digitize existing specimens from the Johns Hopkins Human Temporal Bone Collection; 2) Develop efficient harvesting, rapid histology, and spatial transcriptomics workflows for new specimens and establish partnerships for collecting specimens during autopsies performed on participants in epidemiological studies; 3) Education/outreach activities to encourage other investigators to engage in temporal bone research. The large temporal bone collection and the inner ear illustrations of Max Brödel at Johns Hopkins contributed greatly to our early understanding of many diseases of the ear, such as the discovery of glomus bodies, the histopathologic correlates of presbycusis, and innervation of the saccular maculae. The collection has also shown enduring value, contributing to our understanding of the prevalence of a dehiscent superior semicircular canal and arachnoid granulations as well as the role of melanin in cochlear and vestibular protection. Sharing this valuable collection with other researchers while applying novel techniques in digital processing and education will accelerate advances in and knowledge of otopathology. We look forward to reinvigorating this tradition of excellence by developing a new collection, applying modern techniques, and integrating otopathology with population-based studies that include comprehensive otologic testing and functional measures.

NIH Research Projects · FY 2025 · 2022-09

Functional Magnetic Resonance Imaging (fMRI) shows great promise in characterizing brain circuits and networks related to human mental function and identifying pathophysiological changes underlying mental health disorders, healthy and pathological aging, substance misuse, and beyond. Great strides are being made in many areas, but the vast majority of fMRI research relies on the simplifying assumption of a canonical (or highly constrained) hemodynamic response function (HRF) that is substantially inaccurate. The HRF varies across brain regions, individuals, and age, but estimating it with sufficient accuracy and precision is problematic in small to medium-sized studies. As a result, over 95% of fMRI studies use a canonical HRF of fixed form. This results in substantial bias, power loss, and confounding. These problems apply to both task-based and connectivity studies, which rely implicitly on the assumption of a constant HRF across regions and individuals. In response to the “Notice of Special Interest (NOSI) regarding the Use of Human Connectome [HCP] Data for Secondary Analysis”, propose to use the Lifespan aged 5-100) combined with advanced statistical modeling t we HCP data (n=~3,600 high-quality datasets from people o address this issue. In Aim 1, we will contrast commonly used HRF models across the lifespan based on reliability and ability to ‘decode’ task state and phenotypic variables (e.g., cognitive function and mood). We develop novel methods for extracting meaningful phenotypic information from HRF shape and population inference, and develop robust software for best-in-class models. In Aim 2, we integrate best-in-class HRF models into a novel Gaussian process model and use it derive a demographic-specific, spatiotemporal HRF atlas, providing customized HRFs based on readily measurable characteristics (age, sex, and body- mass index) and brain region. In Aim 3, we use the HRF atlas to deconvolve rs-fMRI data and construct an HRF-corrected connectome map. We validate the HRF models, atlas, and connectome on two independent HCP Disease Connectomes and the CAM- CAN dataset (n=~700), and share the atlas, connectome, and software integrations with the research community. The development of these large-sample models will provide more accurate and precise estimates of task-related fMRI activity and connectivity in basic and clinical studies of mental health, aging, substance use, and beyond.

NIH Research Projects · FY 2025 · 2022-09

This project aims in developing treatments for an atypical Alzheimer's disease (AD) variant, usually affecting the left hemisphere and comprising the logopenic variant primary progressive aphasia (lvPPA), thus, PPA-AD. There are no pharmacological treatments available for PPA, and the only treatment shown to alleviate language deficits is speech-language therapy. Treatment research in AD has emphasized targeting neuronal synaptic transmission. We were amongst the first groups in the world to show the efficacy of a neuromodulation technique that targets synaptic transmission (transcranial direct current stimulation, tDCS) in providing significant symptomatic relief of language impairments in PPA. In the largest-to-date, double-blind, sham-controlled clinical trial we demonstrated the efficacy of tDCS as an adjuvant for speech-language therapy for the treatment of naming and spelling deficits in PPA. However, the efforts to slow language degeneration are hindered by the fact that these individuals also suffer from additional cognitive deficits. This is especially true for individuals with AD etiology (pathology and atrophy distribution). Early in the disease, individuals with PPA-AD present with additional cognitive deficits such as verbal short-term memory impairment, even believed to be a primary underlying cause of language deficits. However, treatment of these deficits has not been investigated in PPA- AD using neuromodulation approaches. To address this gap, the proposed research aims to answer the following question: How can we implement neurostimulation-based treatments to maximally generalize their benefits to vital language/cognitive functions? We will do that by employing: (a) a behavioral therapy that directly targets verbal short-term and working memory (vSTM/WM) deficits and that has been shown to effectively generalize even to untrained language functions in post-stroke aphasia, and, (b) targeted neuro- stimulation (high-definition tDCS) based on recent network-neuroscience and neuro-rehabilitation models. In Aim 1, we will compare the efficacy of tDCS delivered over the left supramarginal gyrus (LSMG) vs. the left dorsolateral prefrontal cortex (LDLPFC), both coupled with vSTM/WM behavioral treatment, specifically examining the generalization of treatment effects to untrained vital language-specific and executive cognitive functions in PPA-AD. In Aim 2, we will implement neuroimaging techniques to understand the mechanisms of tDCS-induced changes in terms of: (a) network functional connectivity, (b) previous and novel metabolites such as GABA and glutathione (related to oxidative stress in neurodegeneration), and (c) blood oxygenation, using perfusion imaging. Finally, in Aim 3, we will evaluate novel predictors of responsiveness to tDCS such as perfusion, sex and sleep, thus complementing our previously identified clinical, neural and behavioral predictors (variant, brain volume and initial language/cognitive performance). A better understanding, based on recent advances in network neuroscience, of how tDCS benefits may generalize to untrained language and executive cognitive functions has the potential to revolutionize the development of effective treatments for PPA-AD.

NIH Research Projects · FY 2025 · 2022-09

Dysfunction of RNA metabolism has emerged to play crucial roles in multiple neurodegeneration diseases, including Alzheimer’s Disease (AD) and Alzheimer’s Disease related dementias (ADRD), such as frontal temporal dementia (FTD). One pathologic hallmark of these diseases is the nuclear clearance and cytosolic aggregation of the RNA binding protein (RBP) TAR DNA binding protein-43 (TDP-43), which is found in 20-60% AD patients and 50% FTD patients. TDP-43 has multiple functions in mRNA processing. It is also implicated in regulating retrotransposon activation, but the molecular mechanism is not resolved. Retrotransposon elements are mobile genetic elements that copy themselves by transcribing into RNA, reverse-transcribed into DNA and then inserted into new sites in the genome, a process known as retrotransposition. Long interspersed nuclear element-1 (LINE1) is the only currently active, autonomous family of retrotransposon elements in human, and accounts for ~20% of the human genome. Only a small subset of LINE1s are thought to be mobile. The majority are inactive due to truncations, rearrangements and point mutations. There are increasing interest in understanding the “noncoding RNA” functions of LINE1 RNA in chromatin state regulation. In this research project, we will decipher the molecular mechanism of LINE1 RNA dysregulation, particularly the dysfunction of LINE1 RNA decay pathway caused by TDP-43 loss of function, which is associated with the pathology of Alzheimer’s Disease and Alzheimer’s Disease Related Dementias. We will determine the causal relationship of LINE1 RNA elevation with chromatin accessibility, histone modification and transcriptional gene network disruption. We will combine human induced pluripotent stem cell-differentiated neurons and postmortem tissues from AD and FTD patients to dissect the molecular mechanisms associated with TDP-43 proteiopathy. Our proposed study will provide deeper mechanistic understanding of the retroelement dysregulation in AD/ADRD and its role in functional genomics, which is largely understudied in the past. The findings will help understanding disease mechanisms and facilitating therapy development for Alzheimer’s Disease and Alzheimer’s Disease Related Dementias.

NIH Research Projects · FY 2025 · 2022-09

Project Summary: Epidemiological and pathological studies have implicated lifestyle, microbial, and environmental factors in prostate cancer etiology/risk. A potential link between these factors and prostate carcinogenesis is the presence of chronic inflammation associated with atrophy (PIA) in prostates of aging men. Yet, there is a paradox surrounding the role of the immune response in prostate cancer: “the inflammation paradox”. On one hand, inflammation may be a driver of carcinogenesis. On the other, the immune system is known to seek and destroy cancer cells. The majority prostate cancer lesions are “immune deserts”, and ICIs are ineffective in most cases. Why is there an evidently strong immune reaction in non- neoplastic regions in PIA, but a lack of a robust immune response in most prostate cancers? We hypothesize that chronic inflammation in PIA represents evidence of an innate immune response that drives carcinogenesis. However, in this inflammatory “proving ground”, only cells that can epigenetically switch off this response can emerge to become aggressive neoplastic precursors. We hypothesize that the paucity of immune infiltrates and lack of PD-L1, is evidence that prostate cancer cells develop a number of different mechanisms that evade anti-tumor adaptive immunity. We postulate that additional cell non- autonomous immune suppressive mechanisms enable disease progression. We propose 3 synergistic Research Projects (2 basic,1 translational) to mechanistically test key questions stemming from our “proving ground” hypothesis. In Proj 1 (Basic Science) we hypothesize that the STING induction in PIA drives acute and chronic inflammation, leading to cell injury/cell death and proliferation. Second, in a subset of PIA cells, epigenetic silencing of STING dampens of the immune response, allowing them to emerge as overt pre- neoplastic cells. We will test this in animal models and in translational studies employing annotated and molecularly characterized prostatectomies. The combination of PTEN loss and MYC copy number gain is an independent predictor of poor outcome in prostate cancer. We hypothesize that the combination of MYC and PTEN stimulates a cell non-autonomous immune evasion mechanism induced by the recruitment of immuno- suppressive myeloid cells, and fibroblast activation protein (FAP)-positive fibroblasts. Proj 2 (Basic Science) will test these hypotheses in animal models and in human tissues. Recently introduced imaging technologies have raised the hypothesis that PET/CT imaging results may be able to predict molecular and tumoral micro- environmental characteristics of aggressive prostate cancer. PET imaging for PSMA using PyL PET/CT has been FDA approved for imaging high risk men prior to prostatectomy. In Proj 3 (Translational) we employ PET/CT scanning for PSMA and combine this with mpMRI to address these hypotheses. Also in Proj 3 we will apply newly developed/developing PET imaging agents to non-invasively and longitudinally study the extent of M2 macrophages and cancer associated fibroblasts in our mouse prostate progression cancer models.

NIH Research Projects · FY 2024 · 2022-09

Project Summary In 2016, faculty from Johns Hopkins medicine invited all academic medical centers in the US to collaborate in creating the High Value Practice Academic Alliance (HVPAA), a multi-institutional, multispecialty organization designed to efficiently and effectively advance value-based quality improvement initiatives on a national scale. As of January 2022, more than 200 faculty leaders from 100 academic centers in the US and Canada serve as institutional or departmental representatives. Members convene on monthly conference calls for information sharing, organize the annual conference, collaborate on multicenter publications, and direct two free year-long high-value care professional development programs (Future Leader Program for trainees and VITAL for early- career faculty). This proposal requests support for the 5-7th annual Architecture of High Value Health Care national conferences. Since its inception, the annual conference has advanced more than 600 value-based quality improvements initiatives and fostered broad collaboration across academic centers. Presentations from the 1st four conferences contributed to a blueprint encompassing five key areas health systems must address to become genuinely high-value medical centers: (1) Diagnostic and therapeutic efficiency and effectiveness, (2) quality-driven care pathways to reduce unwarranted practice variability, (3) Care transitions and in particular hospital discharge, (4) Optimizing patient care setting and improving the caliber of ambulatory care, to reduce avoidable use of the emergency department and hospital, and (5) Preventative medicine to prevent disease and evidence-based screening from reducing late-stage diagnoses. For the following three conferences, we will prioritize equitable delivery of high-value care and interdisciplinary teamwork in addition. Presentations by medical students and trainees are prioritized, as their engagement ensures that value-based care will become the standard for future generations. Our Future Leaders Program bolsters this mission for trainees and the Value Innovation Teaching and Leadership Program (VITAL) for junior faculty. The conference is a critical component of these two programs by fostering a network of high-value care champions and building a foundation for their future success.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Pancreatic cancer arises from precancerous lesions that are curable if detected and treated early enough. Recent multi-region genomic analyses of one type of precancerous pancreatic lesion, intraductal papillary mucinous neoplasm (IPMN), suggest unique evolutionary and selective pressures in IPMNs compared to invasive cancers, underscoring the potential importance of selection in the progression of precancerous lesions. We propose to characterize the role of selective forces in IPMNs using comprehensive molecular analyses and computational data integration of both human IPMN tissue samples and organoid cultures. We will determine the molecular features of progressed subclones in human IPMN samples using multi-region whole genome sequencing and RNA-sequencing. In addition, we will develop and apply novel multi-omics computational methods to integrate DNA and RNA-sequencing data to delineate selective forces in human IPMNs. We will determine the function of progressed clones identified by whole genome DNA sequencing with gene signatures inferred from bulk transcriptional data in a new semi-supervised framework. We will then employ a three-dimensional in vitro organoid culture model of human IPMN cells to characterize the relative contributions of selective pressures over time. We will use combined DNA sequencing and single-cell RNA- sequencing to identify gene expression signatures of progressed subclones in our organoid model, further adapting our computational framework to single cell RNA-sequencing data. Taken together, the proposed studies combine direct analysis of human tissue samples and manipulation of human precancerous cells in three-dimensional culture with novel multi-omics computational integration to greatly expand our knowledge of the role of selection in pancreatic cancer precursor lesions.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Sleep disordered breathing (SDB) is a highly prevalent disease that is associated with increased risk for incident diabetes. Mechanisms of SDB-induced dysglycemia have not been elucidated. Hypoxemia has been implicated as a key pathogenic factor in the development of glucose intolerance and diabetes. High altitude populations, who experience chronic hypoxia, represent unique cohorts to study the role of nocturnal hypoxemia in the pathogenesis of SDB-related diabetes. Our group previously demonstrated that severity of nocturnal hypoxemia, independent of daytime oxyhemoglobin saturation, was associated with elevated hemoglobin A1c. To date, no study has shown that targeting nocturnal hypoxemia improves glucose control. In this application, we will investigate the hypothesis that nocturnal hypoxemia causes dynamic nocturnal glucose elevations, that are mitigated by oxygen. We will employ continuous glucose monitoring to examine the temporal association between glucose and oxygenation dynamics in an observational study and examine the effects of nocturnal oxygen supplementation in a randomized cross-over study. Successful completion of this proposal will provide mechanistic proof that nocturnal hypoxia is a reversible cause of dysglycemia in highlanders. The applicant, Dr. Luu Pham, is a physician in Pulmonary, Critical Care and Sleep Medicine, who seeks to develop a research career focused on the pathogenesis of SDB-related metabolic diseases. This proposal would allow the applicant dedicated time to conduct the outlined research project, as well as pursue didactic training in design and conduct of clinical trials and further quantitative methods relevant to this project and future research plans. The data generated from this research proposal will form the basis for an R01 application to examine the impact of treating of hypoxemia in the pathogenesis diabetes. The career development plan for this applicant includes a structured approach to mentoring, didactic coursework focused on a specific research goal, participation in local and national meetings and identification and regular assessment of career milestones. The research environment provided by Johns Hopkins University as well as the mentorship team described in this application will assist in Dr. Pham’s successful completion of his career and research goals. The Division of Pulmonary and Critical Care Medicine and Johns Hopkins University have a long history of training successful young clinical researchers. The pre-existing high-altitude research infrastructure and trained field staff, as well as the applicant’s established and ongoing collaboration with his team of mentors, will ensure that the study goals will be completed within the timeframe of this award. We have assembled a mentoring team of established faculty with distinct, complementary strengths in high altitude research, clinical trials, metabolic sequelae of SDB and biostatistics, relevant to this proposal. In addition, each member of the mentoring committee serves as an excellent role model for the applicant’s career development into an independent investigator.

NIH Research Projects · FY 2026 · 2022-09

IMAGING MASS SPECTROMETRY-BASED METABOLOMIC ANALYSIS OF THE ALZHEIMER'S BRAIN PROJECT SUMMARY Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Although multiple genes and their mutations linked to AD pathogenesis have been reported, the pathogenic mechanisms of AD still remain elusive. The focus of the majority of AD research has been targeted towards the selective loss of specific neuronal populations. Still, less effort has been spent in understanding reactive astrocytes, a feature common to injury and disease in the aged brain. Recently, we described a subtype of reactive astrocytes that are observed in various human neurodegenerative diseases, including AD and Parkinson's disease (PD). Activation of microglia leads to the conversion of astrocytes into neurotoxic reactive astrocytes via secretion of IL-1, TNF, and C1q. Blocking microglia activation with the drug, NLY01 prevented astrocyte conversion to reactive astrocytes providing neuroprotection. The conversion of resting microglia and astrocytes to reactive ones is deeply related to changes in energy metabolism and lipid composition in the cell. Resting microglia mainly rely on oxidative phosphorylation for energy production. When microglia metabolism converts from oxidative phosphorylation to glycolysis, microglia are activated. This change subsequently converts resting astrocytes to reactive ones ramping up their glucose consumption. Long-chain saturated lipids in APOE and APOJ secreted by astrocytes also have been reported to show neurotoxicity. Therefore, understanding metabolomic changes in microglia, astrocytes, and neurons during the course of neurodegeneration is likely to provide a deeper understanding of AD pathogenesis. Imaging mass spectrometry-based metabolomic analysis will provide a view of cell-type and region-specific metabolomic changes in the brain. To study metabolomic changes in a cell-type-specific manner, we propose three specific aims. (Aim 1) We will examine region- and cell-type-specific metabolomic changes in the microglia-astrocyte-neuron axis in the brain of mice overexpressing amyloid β and in response to pathologic tau. (AIM 2) We will examine region- and cell-type- specific metabolomic changes in control and AD human post-mortem brains. (Aim 3) We will compare region- and cell-type-specific brain metabolomic changes of mice overexpressing amyloid β and in response to pathologic tau with the ones lacking microglial activation by the treatment with PLX3397 or NLY01. The completion of these aims will provide a better understanding of cell-type metabolome dynamics during aging and neurodegeneration mediated by overexpressed amyloid β and pathologic-tau injection. The novel information acquired in this study will provide indispensable insights into cell-type-specific AD pathogenic mechanisms and offer new opportunities to develop new AD treatments targeting microglia and astrocytes. Furthermore, this strategy can be expanded to study the pathogenesis of other brain diseases. 1

NIH Research Projects · FY 2025 · 2022-09

Abstract Our overarching goal is to investigate the impact of early life immune response to a broad array of pathogenic and commensal microbes and exposure to multiple environmental pollutants on the development and prognosis of allergic diseases from birth up to age 18 years and their underlying molecular pathways. Our project is motivated by growing evidence that in-utero and the first few years of life are critical windows for the development of immunity, and by observations that early life exposures to microbes and environmental pollutants may have a profound impact on future risk of allergic diseases. To achieve our goal, we will leverage the rich resources of the Boston Birth Cohort (BBC), with ~3,500 mother-child pairs who were enrolled at birth and followed prospectively. Through our prior research in the BBC, we have established essential clinical, laboratory and computational infrastructures, and obtained longitudinal epidemiological and clinical data, and multi-omics (genome, epigenome, metabolome) data as well as archived biospecimens. We have shown that the BBC is a high-risk population for adverse environmental exposures and the children of the BBC have a high prevalence of immune-related disorders, including food allergies, early childhood recurrent wheezing and childhood asthma. The breadth, depth, and high quality of the BBC data and biorepository have been demonstrated in over 120 peer-reviewed publications. We also have generated compelling preliminary data to support our study aims and hypotheses. Specifically, by including 1,000 mother-child dyads of the BBC with key longitudinal data elements and biospecimens from birth up to age 18 years, we aim to investigate: (1) effects of early life immune response to microbes on child allergic phenotypes; (2) effects of early life exposure to environmental pollutants (e.g., air pollution, toxic metals) on child allergic phenotypes; and (3) molecular pathways underlying the link between early life environmental exposures and allergic diseases. We will harness cutting-edge antibody profiling technology (PhIP-Seq) to profile IgG and IgE antibodies in 1,000 BBC children at three important developmental windows (birth, 1-2 years, and 15-18 years). This will provide deep phenotyping of a child’s antibody profile and identify longitudinal changes in the context of prenatal, perinatal, and postnatal genetic and environmental interactions. Our ability to profile the antibody repertoire and define allergic phenotypes across critical developmental windows allow us to delineate their longitudinal trajectories, temporal and dose-response relationships between the exposures and outcomes. Successful completion of this study will help identify important early life risk and protective factors, along with novel biomarkers for prediction or therapeutic targets. Ultimately, we hope that high-risk newborns can be identified, and effective interventions can be initiated during the earliest developmental windows when they may have the greatest life- long benefit.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT: The proposal in this application outlines a five-year training program for the development of a career as a physician-scientist in neonatal neuroimmunology. Candidate, Dr. Maide Ozen, is an Early Stage Investigator and an Assistant Professor of Pediatrics at the Johns Hopkins University School of Medicine. The proposed research will be carried out under the primary mentorship of Dr. Lauren L. Jantzie, PhD, an expert leader in translational investigations in perinatal brain injury. Candidate’ s co-mentors are Dr. Shenandoah Robinson, M.D. an expert in oligodendrocyte biology and Dr. Frances J. Northington, M.D. an esteemed Neonatologist and a leader in cell death pathways in the brain. Specific training objectives of this K08 proposal are: develop Dr. Ozen’s scientific and professional skills in 1) advanced neuroimmunology and neuroscience techniques, 2) gene-protein interactions specific to Heme oxygenase-1 (HO-1) to lay the foundation for a future R01 application. Neuroscience research at JHU is exceptionally strong. Dr. Ozen and her mentoring committee have developed a focused approach to achieve these objectives. The experiential research environment, intellectual guidance and carefully selected didactic courses, each matched to a specific aim in this proposal, will ensure rigorous training in neuroimmunology for achieving independence. White matter injury (WMI) is the most common form of brain injury in preterm neonates. Key focus of candidates proposed project is interrogating HO-1 pathway, for the first time, in perinatal WMI. HO-1 is an anti- inflammatory, anti-apoptotic and cytoprotective enzyme with essential roles in immune regulation and is expressed in mononuclear cells, microglia and oligodendrocytes. The candidate is the first to show that chorioamnionitis-induced perinatal alterations of HO-1 pathway in the developing rat brain correlate with neuroinflammation. Therefore, utilizing a validated CHORIO model of Cerebral Palsy, the candidate will first determine how CHORIO alters HO-1 pathway in WMI; second, determine if intracellular HO-1 expression in peripheral mononuclear cells and brain microglia differentially contribute to WMI in CHORIO; third, determine if modulation of HO-1 pathway will protect from WMI in CHORIO. Completion of the targeted career development program and research proposed in this proposal will allow the candidate to learn new technical skills and gain neuroscience expertise to excel as a physician-scientist in neuroimmunology and advance the field of HO-1 biology to perinatal brain injury. Rigorous inquiry in regulation of HO-1 pathway in CHORIO could provide discovery of novel biomarkers and targeted therapeutic strategies for perinatal immune-mediated WMI.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Thrombectomy has significantly improved stroke outcomes. Nearly 80% of our clinic population now present with small strokes and low NIH Stroke Scale scores. However, greater than half endorse significant problems with attention, executive function, and processing speed. For many, significant recovery is seen by 6 months, but up to one third experience persistent vascular cognitive impairment. A biomarker to robustly predict who will exhibit long-standing deficits would enable us to initiate early interventions to slow or even prevent decline. Our work with MEG suggests global disruption of cognitive networks irrespective of stroke size or location; however, the compensatory mechanisms that allow some to recover but fail in others are poorly understood. There is a critical need for a noninvasive, inexpensive screening tool that can be widely implemented. The scientific premise of this proposal is two-fold: (i) using MEG and EEG we can determine functional network characteristics affecting both those with transient post-stroke cognitive impairment (psMCI) and persistent vascular cognitive impairment (VCI) as well as the compensatory mechanisms responsible for recovery, and (ii) a novel deep learning model that performs multimodal (MEG and EEG) learning to find shared signatures of VCI, but ultimately yields a model that needs affordable EEG-only data, will yield a powerful biomarker that can predict conversion of psMCI to VCI early after stroke. This proposal will pursue three specific aims. 1) Identify neurophysiologic similarities between transient psMCI and persistent VCI; 2) Identify specific features of functional connectivity that prognosticate conversion to VCI; 3) Design a digital biomarker that predicts conversion using functional brain networks that can be extended from MEG to EEG. To achieve these aims, we will collect both MEG and EEG data from 200 patients with minor stroke, evaluate their signals with expert neurophysiologists, and monitor the patient’s yearly conversion rate to VCI. We will then design and validate a deep learning model called Siamese Multiple Graph to Gauss (SMG2G), which performs multimodal learning on MEG and EEG network (graph) data but ultimately yields a model that needs EEG-only data to make predictions of conversion to VCI. The final product will be an EEG digital biomarker that can be readily measured and widely employed across the country. The research proposed in this application is innovative because it is the first to use functional network signals to design a biomarker for VCI that is inexpensive and widespread, yet robust, and achieves this by cutting edge machine learning. It is also significant because it will advance the field vertically both scientifically and clinically by enabling large-scale, early detection of VCI. Our team is well-prepared to undertake this project, with clinical and engineering expertise, strong collaborations, preliminary data supporting the aims, and institutional support. Patients with minor stroke have significant potential to fully recover. A biomarker to detect the high likelihood of conversion to VCI will allow us to design, implement, and monitor the effectiveness of targeted interventions to slow or even prevent cognitive decline.