Johns Hopkins University

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract The visual accessibility of a space refers to the effectiveness with which vision can be used to travel safely and pursue intended activities in the space. Visual accessibility of a space reduces significantly for impaired vision, leading to higher risks of encountering hazardous situations, falling, and being disoriented in navigation. It is possible to improve the environmental accessibility by enhancing the visibility of informative or hazardous features through cost-effective modifications of contrast, illumination, and materials. However, it is difficult for people with normal vision, even those with rehab expertise, to judge whether specific objects or features would be visible for individuals with different levels of vision impairment, or to examine the efficacy of modifications. This research aims to develop an objective tool for evaluating the accessibility of indoor spaces to complement the current observational practice in visual environmental evaluation. This tool will be implemented as a smartphone app for easy use by rehab specialists and the general public. Using computer vision algorithms, RGB camera and LiDAR sensing technology, the tool will 1) visualize an environment for a specified level of vision impairment, 2) flag hazardous features, such as edges of stairs and chairs that are not visible for this level of vision, and 3) generate visibility metrics that quantify the visibility for a given object to the level of vision. The development and validation of this tool will be conducted in environments with different levels of realism including high-dynamic range images on digital displays, controlled lab spaces simulating real environments, and real clinical and home environments. The engineering approaches will be established through rigorous sensor testing in real environments. The computational algorithms will be developed using a large database of visibility ratings by low vision participants. The app will be first validated in controlled laboratory environments and then implemented in complex real environments including eye clinics and homes. This research strives to support the highest level of independence for people with vision impairment before they need to rely on assistive technology or human assistance. This endeavor is supported by a team with expertise in low vision research and rehabilitation, computer vision and modeling, architecture lighting and design, and assistive technology development. The proposed tool will facilitate the services provided by rehab specialists, caregivers, and facility managers in environmental evaluation and modification which will in turn enhance safety and independence in people with vision impairment. It will also facilitate telehealth by allowing easy sharing of home evaluation results. A tool that provides quantitative measures will contribute to the consciousness-raising of visual accessibility among the public. The engineering and computational approaches, once established, can be extended to platforms other than the smartphone models in the current development phase and can generalize to broader environmental contexts.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT The incidence of pancreatic ductal adenocarcinoma (PDAC) is increasing, and despite the use of conventional therapies, including immune checkpoint inhibitors, 5–year survival remains dismal, at ~12%1. Late detection and therapeutic resistance constitute the two cardinal challenges in PDAC management. However, it is now clear that, following the initial KRAS mutation, it takes over a decade for overt cancer to develop from premalignant lesions, called pancreatic intraepithelial neoplasia (PanIN). Recent studies have also identified individuals at a high risk of PDAC who have a strong family history or harbor pathogenic variants of cancer susceptibility genes. Imaging further identifies premalignant pathologies, such as intrapapillary mucinous neoplasia (IPMN). Both advances––genetic testing and imaging––together with the exceptionally protracted, >10–year–long, window of silent PDAC progression underscore the opportunities to intercept progression. Here, we have targeted mutated KRAS (mKRAS) that drives up to 90% PDACs. We found previously that a KrasG12D vaccine halts PanIN progression in 40% of KPC mice that harbor a heterozygous KrasG12D mutation. This study provided the premise for testing our mKRAS vaccine comprising six peptides corresponding to the most common KRAS mutations in patients with resected PDAC (NCT04117087). We provide evidence for safety, induction of mKRAS–specific predominantly CD4 T cells, and improved disease–free survival. These data prompted us to initiate a study to evaluate the safety and immunogenicity of vaccine in genetically predisposed individuals (NCT05013216, Aim 1). Promising preliminary data establish conceptual and technological feasibility. We hypothesize that the mKRAS vaccine will (a) trigger mKRAS–specific anti–tumor immunity in individuals with premalignant lesions–– PanINs or IPMN––and (b) slow progression of PanINs to PDAC with a survival benefit in KPC mice when given prior to the induction of the KrasG12D mutation. Aim 1 (already initiated) and Aim 2 will study the safety and immunogenicity of mKRAS vaccine in individuals with genetic predisposition and high–risk IPMN, respectively. We will study mKRAS–specific T cells in terms of memory, exhaustion, and polyfunctionality, as well as clonality and richness of the T cell repertoire. In Aim 1, we will also identify TCR clones with cytotoxic gene signatures and validate their function by knocking in the selected TCRs into human T cells. Aim 2 will allow us to examine the effect of vaccine on premalignant IPMN tissue using image mass cytometry. In the spirit of bidirectional translation, we will move back, in Aim 3, to the inducible version of the KPC mouse to determine the optimal timepoints for vaccine interception, characterize longitudinal changes in the immune compartment of PanIN lesions, and investigate the role of CD4 T cells in preventing PanIN–to–PDAC progression. Establishing vaccine effects in high–risk cohorts, together with murine studies on changes in the cellular architecture and evolving immunosuppressive or pro–oncogenic signals, should allow for novel combinatorial strategies in which vaccines would be co–administered with immune modulators to ensure long–term efficacy in high–risk groups.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The discovery of antibiotics greatly decreased the public health burden associated with bacterial infections; however, antibiotics also disrupt the beneficial microbiome. An added challenge is that the efficacy of treatments is often not what is seen in the laboratory setting in part due to the fact bacteria live in mixed microbial populations. While some mechanisms of community protection have been well studied such as degradation or sequestration of the antibiotic, significantly less effort has gone into understanding the ways that physiological changes protect the cells. Efforts to study mixed microbial systems often use synthetic communities where interactions are dominated by nutrient competition and stress response pathways. To address these issues, our laboratory uses a naturally formed community of lactic acid bacteria and Acetobacter that is highly reproducible form natural environments and displays coevolved properties. Recently we have shown that the beneficial microbe Lactiplantibacillus plantarum has a difference in antibiotic sensitivities when grown in a mixed microbial community with Acetobacter species compared to when it is grown as an isolated strain. Previous work has focused on how these microbes engage in cross feeding; however little work has gone into understanding how non-nutritional cues might affect cellular physiology. My research aims to close this gap by understanding how L. plantarum and Acetoabcter coordinate growth to modify physiology and sensitivity to antibiotics. Leveraging the chemically defined media I co-created, I have recapitulated this antibiotic sensitivity change with the addition of the small molecule acetate which is produced by Acetobacter. Additionally, I have seen that acetate stimulates the growth of L. plantarum. This work led me to identify a mutant of L. plantarum that can grow fast without the stimulation of acetate. Through a combination of genetic, metabolomic, and microscopic approaches I will identify the underlying molecular mechanism of the phenotype. This project will identify how a cross-phylum signals affect cellular physiology which may have implications across bacterial species to understanding how antibiotic sensitives are modulated by mixed microbial communities.

NIH Research Projects · FY 2026 · 2024-09

Spatial perception is based on various sensory inputs processed within a widely-distributed cerebral network. Cur- rently it is not known how these multisensory mechanisms and their contributions to spatial orientation are affected by aging. Clinical histories in elderly frequently entail experiences of spatial misperceptions with sudden feelings of imbalance or tilt that can lead to injuries that are highly morbid and costly. Here we combine psychophysics and mathematical modelling to investigate age-related changes in these ‘high order’ mechanisms. We have studied spatial orientation in the context of Bayesian spatial model (BSM), which incorporates the sensory signals that encode the head and eye positions to quantify perceived upright orientation. Accordingly, the signal-to-noise ratio in each sensory modality affects its reliability for integration into an internal (i.e., neural) estimate that contributes to spatial orientation. In this novel approach, sensory contributions are measured directly in psychophysical para- digms. In this proposal, we apply the same approach within the quantitative BSM framework to study changes in spatial orientation with aging. We’ve found individuals with vestibular loss have a bias in spatial orientation related to underestimation of their head position. With aging, there is also sensory loss in the vestibular system that can lead to problems with balance and spatial orientation by affecting how the brain reweights sensory information to compensate for such changes. This reweighting process is important to understand for devising diagnostic tools and rehabilitation/treatment programs preferably at the individual subject level. On this basis, our central hypothesis is that in the process of sensory integration for spatial orientation, older individuals underestimate their eye and head positions, resulting in larger errors of spatial orientation and more postural instability compared with younger individuals. To test this hypothesis, we (i) parse out sensory contributions to spatial orientation in aging, and (ii) examine whether they correspond with age-related vestibular physiologic changes and fall risk. A key vestibular input is from the otoliths that contributes to wide range of neurophysiologic functions such as sensing the head motion with respect to gravity and it also drives the vestibulo-ocular response known as the ocular counter roll (OCR). We have developed a video-oculography measure of OCR (vOCR), which is done with a simple tilt maneu- ver. Here we also examine whether vOCR can be used as a biomarker of age-related changes in spatial orientation, postural stability, and fall risk. Overall, this approach gives us a unique opportunity to apply our current research work towards elucidating sensory contributions to changes in spatial orientation with aging. The key developments in this proposal are identifying distinct changes in spatial orientation and examining a clinical measure of otolith function as potential biomarker to assess and track age-related perceptual and balance functions.

NIH Research Projects · FY 2025 · 2024-09

All eukaryotic organisms share the complex need for intricate control of gene expression according to the needs of the cell. This is achieved largely in part by dynamic, precise positioning of nucleosomes at gene promoters, which share a defined chromatin architecture characterized by a nucleosome depleted region (NDR) surrounded by modified nucleosomes. The boundary of the NDR is defined by an upstream -1 nucleosome and a downstream +1 nucleosome. The gene promoter contains necessary sequence elements that have been well established to control transcription initiation such as transcription factor binding sites, the TATA box, and the necessary transcription start sites. It has been reported that the nucleosome can obstruct these sequence motifs serving as a barrier to transcription machinery and its positioning provides an important role in regulating gene expression. Nucleosomes are positioned by chromatin remodeling enzymes that use energy from ATP hydrolysis for nucleosome translocation. Many gene promoters recruit multiple remodelers from the sub-classes (SWI/SNF, INO80, ISWI, and CHD) and transcription factors that direct the necessary transcription machinery. However, the kinetics of how these multiple factors that can all slide nucleosomes work at the same gene promoter remains elusive with existing methods. This proposal is based on my preliminary data designing a real-time reporter of +1 nucleosome position to elucidate how multiple remodelers coordinate transcription initiation using the model organism Saccharomyces cerevisiae. Here, I propose a time- resolved system to observe the kinetically separable steps of reconstituted +1 nucleosome movement on native sequences in the context of sequence specific and general transcription factor binding. In Aim 1, I will use DNA from the promoter of the inducible gene HIS3 to reconstitute a +1 nucleosome FRET construct that will report +1 nucleosome positioning over time. In Aim 2, I will add purified chromatin remodeling enzymes to my nucleosome construct and measure the temporal window of promoter accessibility caused by multiple, opposing remodelers, testing the hypothesis that the push and pull by different remodelers are responsible for promoter accessibility. Additionally, I will image the same construct in the presence of whole cell extracts that contain all the native cell components for comparison with purified remodelers. In Aim 3, to measure the functional impact of promoter accessibility, I will test the hypothesis that nucleosome dynamics control the binding of sequence specific transcription factor GCN4 and the general transcription factor TFIID. These experiments provide a direct reporter of +1 nucleosome positioning kinetics at single molecule resolution. Overall, this work will provide unprecedented insight into the multifaceted kinetic interplay of major chromatin and transcription components at a eukaryotic gene promoter for the transition from repressed to active chromatin.

NIH Research Projects · FY 2025 · 2024-09

The goal of the proposed fellowship is to prepare the applicant, Lauren Yan, for a career in global mental health research to inform programmatic and policy interventions in low-resource communities, with a particular focus on displaced populations. To this end, the applicant proposes to investigate social determinants of mental health, trauma exposure, and mental distress among adult displaced Syrians in Jordan. The applicant will carry out the proposed research while engaging in individualized mentorship, didactic training, and professional development opportunities targeted towards the following training objectives: 1) advancing skills in latent variable modeling methods; 2) developing expertise in complex causal inference methods; 3) gaining experience in new regions and contexts of humanitarian concern; and 4) engaging in professional development opportunities. These objectives will address gaps in Ms. Yan’s training and will help propel her toward a productive independent research career. The proposed research is highly relevant for public mental health. Social determinants of mental health—the living conditions and non-medical factors that impact mental health and illness—are critical for the well-being of over 100 million displaced people worldwide. These populations face exceptional challenges related to their conditions of daily life, which compound the mental health problems associated with high trauma exposure. While social determinants such as employment, healthcare access, education, social relationships, and housing conditions are promising preventive targets for reducing the global burden of mental disorders, existing studies find heterogenous effects with unclear implications for intervention. There is also a paucity of longitudinal evidence from low-resource settings—where most displaced people reside. Consequently, social determinants’ mechanisms of effect are poorly understood in such contexts. Given these gaps in knowledge, research from low-resource displacement settings is needed to inform intervention approaches that more comprehensively support the mental health of displaced populations. To address these gaps, the proposed research will use data from the Syrian Refugee Life Study to: 1) identify the latent factor structure of displaced Syrians’ social determinants of mental health; 2) estimate the main causal effects of social determinants and trauma exposure on mental distress; and 3) evaluate whether social determinants moderate the relationship between trauma exposure and mental distress. The proposed research aligns with Goals 2 and 3 of NIMH’s Strategic Plan by providing contextually relevant evidence and generalizable methodological approaches for understanding mental illness trajectories and enhancing prevention strategies.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY This is a proposal for a two-year career transition award to study atypical B cells as a novel immunologic and metabolic target to improve vaccine responses in immunosuppressed individuals. The candidate is currently a postdoctoral research fellow at the Johns Hopkins University School of Medicine. The proposal builds on the candidate's extensive experience in vaccine immunology and immunometabolism to understand how metabolic reprogramming of B cells contributes to successful vaccine responses. The COVID-19 pandemic has highlighted the need to develop and rapidly deploy highly immunogenic vaccines. However, insufficient immune responses in immunocompromised individuals have emphasized that novel vaccine formulations may be required for specific populations to overcome immunosuppression. To date, the basic immunology behind successful vaccine responses during immunosuppression has not been well characterized. By dissecting the immunologic and metabolic landscape following vaccination in immunosuppressed individuals, we hope to generate building blocks to reverse engineer vaccines for specific patient populations. Preliminary data from the principal investigator indicates that solid organ transplant recipients, who respond poorly to vaccination due to a variety of immunosuppressive drugs, manage to successfully respond to COVID-19 vaccination by expanding atypical B cells that are reliant on fatty acid oxidation. To further these findings, the principal investigator has proposed a research plan consisting of 3 specific aims. Aim 1 will define populations of immunosuppressed individuals who utilize atypical CD11c+ B cells as a salvage pathway to successful vaccine responses. Aim 2 will assess atypical CD11c+ B cell and T cell interaction to determine specific immunological contexts that drive CD11c+ B cell expansion. Aim 3 will target fatty acid oxidation using vaccine adjuvants to enhance atypical CD11c+ B cell development. Together, these studies will answer how atypical B cells develop in the presence of immunosuppression, what agents we can use to metabolically target them, and who will benefit from vaccine strategies targeting atypical B cells. The principal investigator will also learn new techniques necessary to accomplish the proposed research under the advisory team (Drs. Cox, Bailey, Durbin, Ji, and Pearce), all of whom have pioneering expertise in vaccinology, immunology, immunometabolism, and biostatistics. Importantly, her advisory committee collectively has a very strong track record of training both clinical and postdoctoral fellows who have successfully transitioned into independent investigators at top tier research institutions. She will also engage in and present at national seminars, take coursework on metabolic modeling, clinical vaccinology, Jr. Faculty leadership program, grant-writing seminars, and training on running a laboratory. The outlined career development and research plan will provide the candidate with unique cross-disciplinary skills that will enable her transition to independence and identify promising strategies to improve vaccine responses in immunocompromised individuals.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Tuberculosis (TB) remains a leading cause of global mortality. Treatment is efficacious and low cost for most people, but clinic-based (“passive”) case detection leaves millions of people with TB undiagnosed every year. To accelerate progress against TB, we must bring detection and prevention to communities – but community- based interventions, such as systematic screening and TB preventive treatment (TPT), are resource intensive at scale. Evidence on how to target community-based TB interventions for greatest impact and efficiency is therefore urgently needed. Novel data sources – including aggregated cell phone records, genomic sequencing, and empiric estimates of cost – are emerging as critical tools for understanding and fighting TB in high-burden settings. Scientists who can analyze these data using rigorous and reproducible techniques while applying a health policy lens will be essential to our future success against the world’s leading infectious killer. The goal of this project is to identify ways to make community-level TB screening and prevention more impactful and cost-effective by better characterizing the spatial dynamics of TB and Mycobacterium tuberculosis (Mtb) transmission. My training aims include: (1) develop expertise in spatial epidemiology and the analysis of mobility data; (2) obtain training in molecular epidemiology; and (3) gain skills to link these data sources to mathematical models. These aims link to three research aims: (1) refine estimates of subnational TB prevalence by synthesizing emerging sources of data; (2) use genomic data to identify key populations among whom Mtb transmission is concentrated; and (3) project the impact and cost-effectiveness of targeted TB screening and prevention approaches. This research will leverage both aggregated cell phone mobility data and genomic, spatial, mobility, and epidemiologic data from three studies in Uganda and South Africa with unique designs that lend themselves well toward describing the spatial scale of Mtb transmission. To achieve success as an independent investigator, I also plan to obtain experience with field-based epidemiological studies and gain familiarity with the clinical management of TB in high-burden settings. I am a junior faculty member in the Division of Infectious Diseases at the Johns Hopkins School of Medicine with a quantitative background in infectious disease modeling and health policy. My long-term career goal is to become an independent researcher who informs more evidence-based TB policymaking by integrating diverse sources of data into mathematical models. During this award period, I will be mentored by a team whose expertise spans TB epidemiology and clinical care, molecular epidemiology, infectious disease dynamics, human mobility, and mechanistic and statistical disease modeling. Collectively, this research and career development plan will provide a pathway to a career as an independent investigator situated at the intersection of “big data”, disease modeling, and policy analysis who generates evidence that can help reduce the immense and persistent global burden of TB.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY: People with HIV (PWH) experience a heightened risk of atherosclerotic cardiovascular disease (ASCVD) that is attributed, in part, to chronic inflammation. Lipoprotein(a) (Lp[a]), a non-traditional ASCVD risk factor is the major carrier of oxidized phospholipids, which are increased in pro-inflammatory states and which damage vascular cells at the vessel//lumen interface. Identifying Lp(a) risk thresholds, the increment in ASCVD risk for a given increment in Lp(a), would improve risk prediction and inform entry criteria and treatment goals for future Lp(a) lowering therapies in PWH. Our project defines Lp(a) risk thresholds in PWH, explores two possible responsible mechanisms for Lp(a) risk in PWH, and studies regulation of hepatic Lp(a) production in PWH through the inflammatory cytokine IL-6 signaling pathway using sera from PWH. The study results will address critical knowledge gaps and test specific hypotheses related to the Lp(a) associated heightened ASCVD risk in PWH. Aim One utilizes serial coronary and carotid studies of subclinical ASCVD in the MACS/WIHS Combined Cohort Study (MWCCS) cohorts and biospecimens to establish Lp(a)-specific ASCVD risk thresholds in PWH. We will also explore racial disparities as Lp(a) levels in healthy Black people are higher than in healthy White people. Aim Two explores two mechanisms that may explain an association between Lp(a) and the HIV-specific heightened ASCVD risk. The first examines whether Lp(a) is associated with impaired coronary endothelial vascular function, a driver of ASCVD. It uses non-invasive MRI to assess endothelial-dependent coronary function in newly recruited PWH and PWoH. The second measures the association of Lp(a) with coronary vascular inflammation, another driver of ASCVD pathogenesis, using the validated fat attenuation index developed at Oxford University to measure coronary inflammation from MWCCS coronary CT scans. Aim Three examines regulation of Lp(a) production in PWH, a poorly understood aspect of ASCVD risk in PWH. Our preliminary in vitro cell data suggest that the inflammatory cytokine IL-6, known to be elevated in PWH and associated with ASCVD, enhances hepatocyte LPA transcriptional activity via the JAK-STAT pathway. Moreover, the genetic variant rs56393506, known to amplify Lp(a) production, will be studied. We will utilize induced pluripotent stem cell-derived hepatocytes exposed to sera from PWH to examine IL-6 signaling pathways of hepatic Lp(a) production. Additionally, we will utilize the exisitng MWCCS genomics dataset to assess whether known variants linked to Lp(a) identified in published GWAS are associated with the higher Lp(a) levels in PWH. In summary, this comprehensive research identifies Lp(a)-specific ASCVD risk thresholds in PWH by leveraging data from the MWCCS cohorts, studies two mechanisms that may explain why Lp(a) is associated with this risk, as well as pathways of hepatic regulation of Lp(a) production in PWH. We expect the results will increase understanding of the role of Lp(a) in the HIV-specific heightened ASCVD risk and inform Lp(a) treatment goals with Lp(a) directed therapies in this high risk patient population.

NIH Research Projects · FY 2026 · 2024-09

The adverse effects of poor treatment in healthcare settings on cognitive impairment are poorly understood, particularly among aging Americans. My F99 research established an association between these experiences and adverse cardiovascular events. Cardiovascular events are themselves known risk factors for cognitive decline. Building on this foundation, my K00 research will investigate the longitudinal impact of poor treatment in the healthcare settings on cognitive impairment and incident Alzheimer’s disease and related dementias (AD/ADRD). The research will leverage longitudinal data from the Health and Retirement Study (HRS) to test my central hypothesis that poor treatment in healthcare settings increases AD/ADRD risk and risk of cognitive impairment among aging adults. To evaluate this, the K00 research phase will use rigorous statistical methods, including survival analysis to estimate the impact of patient-provider interactions on incident AD/ADRD and longitudinal models to examine how trajectories of cognitive impairment are associated with these experiences over time. To achieve these research goals and facilitate my transition to independence, I will undertake a structured, mentored training program at Johns Hopkins University under the guidance of my primary mentor, Dr. Roland J. Thorpe, Jr.. This plan is designed to help me acquire foundational knowledge in the determinants of cognitive impairment, develop expertise in advanced longitudinal data analysis, and cultivate the leadership capabilities essential for an interdisciplinary research career. The findings from this K00 project will provide preliminary data for a subsequent career development award application, such as a K99/R00 or K01, which I plan to develop during the award period. This research and training trajectory is crucial for my overarching career objective to become a leading researcher focused on barriers to care that impact the cognitive health of aging Americans and aligns directly with the NIA's mission to understand and improve healthcare quality while aging.

NIH Research Projects · FY 2024 · 2024-09

Summary Among all different types of DNA damages, double strand breaks (DSBs) are viewed as the most toxic ones that lead to genome instability. They are created by either endogenous agents such as reactive oxygen species, or exogenous ionizing radiation and chemicals. Unrepaired DSBs drive apoptosis and senescence, and incorrect DSB repair can lead to undesired genome rearrangements, such as deletions, translocations, and fusions. Non-homologous end-joining (NHEJ) pathway, in which the two broken DNA ends are directly ligated without referring to a homologous template, is the primary DSB repair pathway that remains active throughout the cell cycle. NHEJ is also responsible for the assembly of gene segments in V(D)J recombination, where various immunoglobulin genes are generated by exon recombination in immune cells. NHEJ is initialized by Ku heterodimer (Ku70/80) recognizing DSB ends. Upon recognizing a dsDNA broken end, Ku70/80 recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and assembles into the so-called DNA-PK holoenzyme. Other evolutionarily conserved NHEJ factors, including components of the ligase complex (DNA ligase IV, XRCC4 and XLF) are then recruited to the end reparation site. Successful DSB repair through NHEJ relies on the efficient bridging of two broken DNA ends, and this proposal aims to investigate the mechanism of NHEJ by directly visualizing the key steps of repair using single-particle cryo-EM. A more refined picture of the system specifically recognizing and correcting DSBs will provide an unprecedented, comprehensive view of these essential molecular machines during operation, and could lead to the development of novel treatments for various types of human cancer.

NIH Research Projects · FY 2024 · 2024-09

Modified Project Summary/Abstract Section The liver is central to mammalian metabolism and plays a critical role in providing fuel to other tissues particularly when food is limiting. People with disparate inborn errors in mitochondrial fatty acid β-oxidation exhibit life-threatening hypoketotic-hypoglycemia following a fast due to the critical role of fatty acid oxidation to gluconeogenesis and ketogenesis. To understand the contribution of hepatic fatty acid oxidation to systemic metabolic dysfunction, we have generated multiple transgenic mice with an altered ability to oxidize long chain fatty acids via mitochondrial β-oxidation specifically in hepatocytes. Here we will leverage extensive genetic models to understand the contribution of fatty acid oxidation to hepatic and extrahepatic regulation of hepatic and systemic metabolic homeostasis. The expectation is that our proposed studies will describe novel requirements and signaling roles of hepatic fatty acid oxidation that impact the development of obesity and glucose intolerance.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Ovarian cancer is the deadliest gynecological cancer, in part because there is no widely used screening test and 80% of tumors are diagnosed at a late stage. Though ovarian cancer screening has the poten>al to diagnose tumors at early stages when pa>ent outcomes are far be?er (90% survival for stage I cancer vs. <20% for stage III/IV), recent screening trials of protein biomarkers have failed to show a mortality benefit. This has highlighted three main challenges in ovarian cancer screening: (1) Avoiding false posi>ve results leading to unnecessary procedures in a disease with low popula>on prevalence, (2) Achieving high enough sensi>vity to be a clinically useful test, and (3) Ensuring accessibility and pa>ent compliance with screening. Much of my early PhD work, and work in my mentor’s lab, has pioneered high performance blood tests that use cell-free DNA fragmenta>on pa?erns to detect cancer. These fragmentomic assays can detect subtle changes to DNA shed from tumor cells even using low-coverage sequencing of <1mL of blood, and their non-invasive nature makes them accessible and appealing to pa>ents. Given the cri7cal clinical need, the overarching goal of my work is to advance our understanding of ovarian tumorigenesis and develop an accessible, high-performance liquid biopsy for ovarian cancer screening. A limita>on of current liquid biopsies is that they don’t incorporate informa>on from the ~50% of the genome comprising repeat elements, which have long been implicated in carcinogenesis but have been difficult to study due to incomplete genome references and technical genome alignment challenges. In preliminary work, I show a novel approach I developed that allows the study of repeat landscapes in short-read sequencing and that has iden>fied over 800 new elements not previously implicated in cancer. The ability to use this approach in short-read sequencing opens the door to studying repeat landscapes in cell-free DNA, where most fragments are 150-180bp in length. I propose three specific aims: In Aim 1, I will expand this approach to develop novel bioinforma>cs approaches enabling the study of repeat elements in cell-free DNA. In Aim 2, I will develop and test sensi>ve and specific cell-free DNA liquid biopsies for ovarian cancer. In Aim 3, I will characterize genomic changes to repeat elements during ovarian tumorigenesis, from normal >ssue to pre-cancerous lesion to tumor. Successful execu7on of these aims has the poten7al to advance our understanding of the role of the so-called “dark genome” in ovarian tumorigenesis, change the paradigm for liquid biopsies by illumina7ng cancer-related changes to repeat elements, and apply these discoveries to a screening technology for ovarian cancer. This work may also serve as a basis for future pan-cancer screening methods. The proposed work will provide me with outstanding mentorship and scien>fic training in basic and transla>onal science and help me advance my career as a future physician-scien>st and independent inves>gator.

NSF Awards · FY 2024 · 2024-09

NSF FAIROS Research Coordination Networks (RCNs) work to build and enhance coordination of researchers and other stakeholders advancing Findable, Accessible, Interoperable, and Reusable (FAIR) data principles and Open Science (OS) practices. The initial cohort of 10 awards span a wide range of disciplines and have advanced FAIR-OS efforts as well as convened RCN-specific events. To broaden their impact and focus future directions, it is an appropriate time to take stock of RCN efforts and work together to both contextualize successes and clarify gaps as needed to plan sustained efforts in FAIR and Open Science across the US Science research landscape. This PI Meeting will support a community-driven and community-focused effort to help define the role that federal agencies can play in accelerating the pace of FAIROS. The workshop will bring together the research teams in their varied stages of maturity to encourage and support those in their early years while providing new ideas for more mature efforts facing persistent gaps. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Our research program seeks to develop innovative technologies to investigate the role of ADP-ribosylation, an under-studied DNA modification implicated in critical biological processes, including DNA repair, genome replication, and pathogen defense. Though prevalent across all kingdoms of life, from bacteria to humans, the precise function and extent of DNA ADP-ribosylation remain elusive. Current knowledge largely stems from studies on the bacterial DarT-DarG toxin-antitoxin system, and more recently, the discoveries of DNA modification by the anticancer drug target PARP1 in human cells. One significant challenge in the field is the absence of technology capable of identifying DNA ADP-ribosylation sites across the whole genome. Past efforts have involved meticulous mutagenesis using radioactive labeling on predefined oligo sequences or breaking down the genome to individual bases for modification assessment. However, these methods are limited in their ability to provide the broader genomic context of DNA ADP-ribosylation. Our goal is to bridge this gap through the development of two complementary techniques, using the well- characterized DarT-mediated ADP-ribosylation as our model. The first repurposes tools we have developed to study protein ADP-ribosylation and applies them to DNA, using high-throughput Illumina-based sequencing to pinpoint the modification site. The second approach involves nanopore sequencing, using a modified transmembrane protein as both a channel for DNA passage and a biosensor for base identification. By monitoring changes in electric current, we can distinguish specific bases and detect DNA modifications. To optimize this method, we will collaborate with leading nanopore epigenetics expert Dr. Winston Timp at Hopkins and adapt established machine learning approaches for electric current data analyses. We will test and refine our methods using DarT-modified oligos with defined sequences and genomic DNA standards. In summary, this program aims to unravel the complexities of DNA ADP-ribosylation using cutting-edge sequencing methods. These strategies will enable the investigation of the sequence context of ADP- ribosylation sites and, in due course (beyond the current scope), the study of motifs from diverse pathogen toxins and other modifying enzymes such as human PARPs. By developing tools to reveal the mechanisms of DNA repair, gene regulation, and pathogen defense, we anticipate that our work may catalyze significant advancements in healthcare, potentially leading to the development of new treatment and detection tools for a range of diseases, from infections to cancers.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Idiopathic inflammatory myopathies (IIM) comprise a heterogeneous group of chronic autoimmune diseases that can lead to severe muscle weakness and chronic disability; however, it is their association with contemporaneous cancer (cancer-associated myositis, CAM), that is responsible for mortality rates approaching 50%. Myositis-specific autoantibodies have emerged as important biomarkers, and are associated with an increased risk of cancer, but are of limited utility in predicting if cancer will emerge in an individual patient – the majority of patients with the highest-risk antibodies for CAM never have cancer diagnosed. Despite multiple studies focused on the myositis-cancer association, the ability to predict cancer, and the biologic relationship between cancer and myositis, remain poorly understood. Given the inability to predict cancer, the current standard is that the majority of patients with IIM are aggressively screened for cancer for multiple years following diagnosis, leading to increased cost, radiation, and false positive results. Beyond CAM prediction, the biologic relationship between IIM and cancer has been historically neglected; specifically, how cancer emergence and elimination impact IIM outcomes is largely unknown. However, insights gained from the careful study of cancer emergence/elimination in IIM can inform research into disease mechanism, particularly given that cancer has been hypothesized to be responsible for IIM development – so called “cancer-induced autoimmunity”. In this setting, the recent development of highly sensitive, novel cancer detection technologies have enabled the robust study of cancer in IIM cohorts. The overall goals of this proposal are to (i) optimize the prediction of CAM at the individual patient level and (ii) determine how the emergence and elimination of cancer influence IIM clinical outcomes. In Aim 1, we will utilize three of the largest cohorts of IIM patients in the United States to develop and validate a cancer prediction model to inform clinical decision-making. In Aim 2, the relationship between cancer emergence, cancer elimination, and IIM outcomes will be determined using novel liquid biopsy cancer detection technologies, providing insights into IIM pathogenesis and improvements in disease monitoring. Understanding the cancer-IIM relationship will ultimately enable development of novel, effective treatment strategies that are focused on cancer detection/elimination rather than the currently used non-specific immunosuppression approaches.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Our project focuses on the high prevalence of Alzheimer's disease and related dementias (ADRD) among the Hispanic population in the United States. In 2014 ADRD affected nearly half a million Hispanic adults aged 65+ (12% of Hispanics in this age group), and this number is expected to double by 2030. Because ADRD has limited effective interventions, we are mainly concerned with two significant risk factors: low language acculturation and hearing loss. Low language acculturation affects over 60 million Hispanics, hindering their participation in cognitively stimulating activities and potentially accelerating cognitive decline. Hearing loss has recently been identified as a modifiable risk factor for ADRD. Hearing loss is associated with cognitive function and structural brain outcomes that may be early indicators of ADRD. Notably, hearing aids have proven to slow cognitive decline among older at higher risk for ADRD. Despite this, few Hispanic adults with hearing loss use hearing aids. Our research aims to investigate the potential effect modification of low language acculturation on the association between hearing loss, and cognitive function in Hispanic adults. Using data from two studies of Hispanic adults—the Hispanic Community and Health Study/Study of Latinos (HCHS/SOL), and the Northern Manhattan Study of Metabolism and Mind— we have three specific aims: Aim 1. Examine whether the association between hearing loss and cognitive decline over approximately six years among Hispanic adults in the U.S. differs by levels of language acculturation. Aim 2. Investigate the association between hearing loss and brain health markers on MRI scans and whether language acculturation moderates these associations. Aim 3. Explore the link between hearing loss and Alzheimer's disease pathology markers in a different sample from the NOMEM study and assess the role of language acculturation. Our research will inform public health efforts and raise awareness about interventions that can mitigate the effects of hearing loss and cognitive decline in the Hispanic community. It will have a significant impact on healthcare providers, community organizations, and families, helping them address communication barriers and improve access to necessary support systems.

NIH Research Projects · FY 2026 · 2024-09

Abstract: Thyroid hormone is among the most common prescriptions in the United States. Up to 20% of those on therapy are on doses which suppress the normal pituitary feedback system and result in a low thyrotropin (TSH) level, a sign of excess hormone. We have recently found that a low TSH is associated with an increased risk of a later diagnosis of cognitive disorder. This proposal aims to understand whether excess thyroid hormone is a preventable risk factor for Alzheimer's disease and related dementias. We will look at the relationship over time between thyroid hormone therapy, hormone levels, and dementia in several different large groups of patients. Our goals include identifying those who may be particularly vulnerable to excess thyroid hormone. This work is important because thyroid hormone is frequently prescribed to those with dementia and, with millions of older adults at risk, even simple changes to appropriately prescribe thyroid hormone could have important public health benefits.

NIH Research Projects · FY 2024 · 2024-09

As DNA sequencing becomes faster and cheaper, utilization of whole exome sequencing (ES) and whole genome sequencing (GS) as a diagnostic tool has become widespread. It is essential that physicians understand when these tests should be ordered, how to interpret and use the test results including variants of uncertain significance and secondary findings, and understand the ethical, legal and social issues generated by these results. Recent surveys of medical students at Johns Hopkins University School of Medicine (JHUSOM) revealed a desire to learn more about genetic testing and its broad application to clinical medicine. To this end, we developed the Genomic Medicine Translational Science course (Genomic Medicine TS course) for the 3rd and 4th year JHUSOM medical students. This active learning course consists of three 5-hour days during intersession of the academic years with the objectives of improving the medical students’ understanding of ES and GS. From 2017 to 2022, 296 students took the Genomic Medicine elective. To achieve the course objectives, we developed (and made freely available) the PhenoDB Teaching Tool with accompanying phenotypic and ES data to be analyzed as proband only, in a family setting or cohorts of patients with a particular phenotype. Since 2015, we have used the Genomic Medicine TS course curriculum, tools and resources to teach in the annual McKusick Short Course offered collaboratively by the Jackson Laboratory and JHUSOM. In 2021, with the Brazilian Society of Medical Genetics, we used these materials to teach >350 participants from South America and Europe. In 2022, we guided the faculty at UNIFESP on implementing the Genomic Medicine course curriculum to teach 36 medical and graduate students and postdocs in that Institution. A much more in-depth Genomic Medicine education, as provided by a M.S. degree, would prepare the trainees for careers analyzing the human genome in academic research centers, government, and industry, including biotechnology firms, pharmaceutical companies, and clinical laboratories. It would also help clinical guideline developers provide evidence-based genetic/genomic interpretation to properly use genomic information for patient health care decision-making. Thus, there is a great need for master’s education programs that provide health professional and related trainees with the knowledge and skills required to manipulate, annotate, and interpret human genome data, and a foundation for pursuing genomics research. To address this need, we will leverage our evolving expertise in analysis of genomic data and education (Nara Sobreira, Joann Bodurtha, David Valle, Weiyi Mu, Nancy Hueppchen, Kim Doheny, Winston Timp, Xiao Peng, Jim Stevenson, Dane Witmer, Carolyn Applegate) and the unique tools and resources available at JHUSOM such as the ones offered by the Office of Online Education (Robert Kearns), by the Johns Hopkins Advanced Academic Programs (Robert Lessick), and PhenoDB Teaching Tool to develop a Genomic Medicine M.S. program that will address the expanding need for genomic literacy and create opportunities for these individuals to train others.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Pulmonary endothelial cells (ECs) are in direct contact with laminar blood flow, resulting in exposure to shear stress. Normal blood flow provides a physiologic degree of shear stress at which ECs achieve quiescence. Pathologic changes in shear stress can occur in several conditions ranging from pulmonary embolism, where shear stress acutely decreases due to vessel occlusion, to pulmonary hypertension (PH), where shear stress in the distal arteries increases due to luminal narrowing. These disease entities carry considerable morbidity and mortality despite available therapeutics. The biochemical derangements that occur when shear stress is altered are not well-characterized, and elucidating these pathways may provide novel insight into potential therapeutic targets to prevent long-term dysfunction of ECs. In vitro culture of ECs is often performed under static conditions, leading to underappreciation of the effects of physiologic shear stress on normal cellular function as well as the biochemical and functional impact of shear perturbations. Aquaporin 1 (AQP1), a ubiquitous protein that forms water channels, is known to be expressed in vivo in the pulmonary endothelium, but we noted that AQP1 expression is not observed in human lung microvascular endothelial cells (hLMVECs) grown in static cell culture. My preliminary data show restored AQP1 expression in cultured hLMVECs with exposure to physiologic shear stress, suggesting a critical role of shear stress in dynamically regulating AQP1 expression. Increased AQP1 has recently been linked to important cellular functions, including angiogenesis and proliferation in certain malignancies, as well as contributing to vascular remodeling through apoptosis resistance and hyperproliferation in the ECs from rat models of PH. Regulation of AQP1 is not well-described in hLMVECs but is calcium- dependent in pulmonary vascular smooth muscle cells. Aim 1 of this proposal is designed to elucidate the biochemical signaling that occurs in response to changes in shear stress. In preliminary data, I show intracellular calcium levels increase in response to increased shear stress. I seek to define this signaling pathway focusing on the role of activation of the membrane ion channel, TRPV4, which can increase calcium influx in ECs in response to mechanical stimuli, in regulating AQP1 levels. Aim 2 will explore the functional outcome of changes in AQP1 expression in response to varying degrees of shear stress, focusing on apoptosis and proliferation. Techniques utilized will include but are not limited to cell culture under shear stress, ratiometric calcium measurement, protein and mRNA measurement, immunofluorescence microscopy, and measures of apoptosis and proliferation. Completion of this project will provide novel insight into the impact of shear stress on EC function, and how derangements in shear stress may alter cell signaling and cell growth and survival. The skills acquired in the design and execution of this study and the experimental results obtained will provide the necessary foundation for a K award and an excellent platform on which to start a career as an independently funded clinician-scientist focused on diseases of endothelial dysfunction.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Many important cellular processes are regulated through the formation and dissolution of the biomolecular condensates via liquid-liquid phase separation (LLPS). LLPS enriches specific factors in the biomolecular con- densates while excluding others, thereby creating a unique environment that either promotes or restricts certain biochemical reactions. To investigate how the dynamic process of LLPS and the reverse process that results in the dissolution of biomolecular condensates, biosensors capable of survey the biophysical properties of conden- sates as they form and dissolve within cells are highly desirable. The stability of a biomolecular condensate depends on electrostatic forces as well as hydrophobic interactions between the molecules residing in the con- densate. Currently there are no known biosensors for real-time monitoring of environmental hydrophobicity in living cells, limiting our understanding of how hydrophobicity changes over the lifetime of biomolecular conden- sates. Here we propose to develop a genetically encoded hydrophobicity biosensor, consisting of a pair of fluo- rescent proteins that can undergo Förster Resonance Energy Transfer (FRET). This hydrophobicity sensor will report FRET efficiency as the readout of hydrophobicity value. As proof of concept, we will create a recombinant protein in which the fluorescent profiting pair is fused with paxillin, an important protein in neurite growth, migra- tion of neuron and microglial cell, as well as endocytosis in cells of the neural system. We plan to first establish a calibration curve by which hydrophobicity can be quantified. Then we plan to demonstrate that this hydropho- bicity sensor can be used intracellularly to monitor the hydrophobicity changes in biomolecular condensates to which paxillin partitions. If successful, our design principle can be readily applied to measure hydrophobicity of condensates containing other molecules.

NSF Awards · FY 2024 · 2024-09

The 30th International Conference on DNA Computing and Molecular Programming (DNA30) is the premier forum where scientists come together to advance molecular-scale engineering and biology. This interdisciplinary conference bridges computation, biology, and nanotechnology and attracts top researchers in the fields of computer science, mathematics, chemistry, molecular biology, and engineering. This support will give a new generation of molecular programming researchers the opportunity to present their work and interact with students and senior researchers from other institutions. The scope of topics include: control of molecular folding and self-assembly of nano- and micro-structures; demonstration of biomolecular switches and circuits that process chemical information in vitro and in cells; molecular motors and molecular robots; studies of fault-tolerance and error correction in molecular self-assembly and molecular computation; synthetic biology and molecular evolution; DNA data storage; and software tools for analysis, simulation, and design of molecular structures and circuits. These topics have applications spanning engineering, physics, chemistry, biology, medicine, and education. DNA30 will be held at Johns Hopkins University in Baltimore, MD on September 16-20, 2024. Conference organizers will support travel of up to 25 students and post-doctoral researchers who are either US citizens or enrolled at US institutions. Trainees who plan to present their work at the conference will be prioritized for awards and especially those from institutions that would otherwise be unable to afford conference attendance and members of groups underrepresented in STEM research. The awards will support registration, travel, and accommodations for students. The availability of this support will foster diverse conference attendance, fostering the unique perspectives required to advance human knowledge and develop new technologies. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Intimate partner violence (IPV) and depression are substantial public health challenges for U.S. adolescents, and evidence suggests a clear link between IPV experiences and subsequent depression. Despite their co- occurrence and grave implications, research has not sufficiently explored the nuances and longitudinal patterns of these experiences. The proposed research study addresses the intersecting crises of IPV and depression, focusing on adolescents' unique vulnerability and the consequences of these issues for their short- and long- term well-being. This study leverages the nationally representative NEXT Generation Health Study (NEXT) dataset, which spans seven years of longitudinal data collected annually (2010–2017) from a diverse sample of 2,780 adolescents aged 15 through 23. The study examines the longitudinal relationship between IPV and depression via two specific aims. Aim 1) To identify and characterize subgroups of IPV experience from mid- to late-adolescence for females and males separately. Using Latent Class Analysis, this aim seeks to reveal distinct patterns of IPV experience during adolescence using the first four waves of the NEXT, which measure various types of IPV, including verbal, psychological, physical, and sexual. Subsequently, various background indicators (e.g., age, race/ethnicity, sexual orientation, household structure) will be explored as predictors of subgroup membership. Because previous research points to sex-specific differences in IPV experience, these analyses will be conducted separately for females and males. Aim 2) To examine depression symptom trajectories and the relationship between IPV subgroups and depression trajectories from mid-adolescence to young adulthood. Using dual trajectory modeling, this aim investigates longitudinal trajectories of depression for the full NEXT sample, and then within identified IPV subgroups, exploring variations in onset, duration, and severity of depression symptoms. It is hypothesized that individuals in subgroups described by more severe, persistent IPV will exhibit higher, more persistent depression trajectories over time compared to subgroups described by less persistent IPV experiences, with background indicators influencing these relationships. This study addresses a major gap in knowledge of the longitudinal relationship between adolescents’ IPV experiences and subsequent depression over longer time periods during the critical transition from adolescence to young adulthood, reflecting multiple NIH research priorities. Responsive to the NICHD Child Development and Behavior Branch’s strategic plan theme of improving child and adolescent health and the transition to adulthood and NIMH’s priority for mental health disparity research, this study will improve understanding of IPV and depression among adolescents as they transition to young adulthood. Findings are valuable for tailoring interventions and policies, as different subgroups may have unique risk factors or needs during this key transition period that has important implications for adolescents’ future education, work, and well-being.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY: This is a submission for a National Institutes of Health R01 award called The BLAAST Project, which aims to conduct a randomized, double-blinded, placebo, non-inferiority trial of pediatric care enhanced by a novel automated digital stethoscope, compared to standard care, in Bangladesh (Aim 1) with an integrated implementation assessment (Aim 2) and economic evaluation (Aim 3) over three years among young, low-risk children with non-severe clinical pneumonia. Antibiotics are a mainstay of the treatment of acute lower respiratory infections in young children in low- and middle-income countries (LMICs) like Bangladesh even though most episodes are caused by self-limiting viruses. Innovative child friendly tools that improve the diagnosis of respiratory illnesses, safely reduce the unnecessary use of antibiotics, and are suitable for implementation in LMICs are urgently required to safely improve antibiotic stewardship and stem the rising rates of antibiotic resistance globally. In this project (Bangladesh Lung Auscultation Artificial Intelligence for Antibiotic Stewardship or BLAAST) we aim to utilize a novel FDA-approved digital stethoscope with automated lung sound analytics developed and validated over a period of ten years from evidence across seven LMICs. In Aim 1, we will determine whether treatment failure frequency among children in rural Bangladesh managed by clinical guidelines enhanced by a novel automated digital stethoscope is non-inferior to guidelines alone. We hypothesize treatment failure frequency among `enhanced IMCI' participants will be no worse than standard care by a +/-2% margin, safely reducing antibiotic use by 50-60%. In Aim 2 we will assess digital auscultation implementation and antibiotic use during pediatric respiratory care in rural Bangladesh to inform strategies of antibiotic stewardship. Lastly, in Aim 3 we will evaluate if a diagnostic strategy enhanced by an automated digital stethoscope is a sustainable alternative to standard care for children in rural Bangladesh. We hypothesize that care augmented by a digital stethoscope will have additional benefits via reduced antibiotic use that will outweigh digital auscultation costs resulting in cost-effectiveness compared to current practice. BLAAST affords a unique opportunity to evaluate the efficacy of clinical guidelines enhanced by an automated digital stethoscope on child pneumonia outcomes in Bangladesh, if digital auscultation may be instrumental in the wider antibiotic stewardship strategy, and whether a digital stethoscope diagnostic tool is cost-effective in the care of children with respiratory illnesses.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY In the past, nearly all clinical and research proton magnetic resonance spectroscopy (MRS) studies of human brain metabolism have focused on the resonances upfield (UF) from the water signal. However, over the last few years it has been demonstrated that there are signals downfield (DF) from water that can be visualized using appropriate techniques, which may contain important metabolic information. We have recently shown that it is possible to map these signals throughout the human brain at 3T using 3D magnetic resonance spectroscopic imaging (MRSI). Furthermore, the clinical applicability of this technique would be significantly enhanced if it were possible to simultaneously map both UF and DF peaks at the same time. In this proposal, a new pulse sequence will be developed for DF-MRSI which will have improved sensitivity and excitation profiles compared to our current approach. Rigor and reproducibility will be carefully assessed; the sensitivity and reproducibility of this new approach will be compared to our previously developed DF-MRSI methodology in 10 healthy subjects scanned at two time points. In addition, assignment of DF metabolite peaks will be investigated in detail using multiple metabolite solutions scanned under carefully controlled physiological conditions. This novel pulse sequence may also be used for UF-MRSI acquisitions. Reproducibility and congruence with conventional UF-MRSI acquisition methods will be established, again by the study of 10 healthy volunteers at two time points. Finally, simultaneous UF- and DF-MRSI will be implemented with optimal sensitivity for both sides of the spectrum. Again, sensitivity and reproducibility will be compared to sequential UF- and DF-MRSI measurements made in the same total scan time in 10 healthy subjects at two time points. In parallel with the pulse sequence development and data acquisition aims, we will also continue to develop and disseminate our MRSI post-processing methods as part of our open source ‘Osprey’ software package, which is available to the clinical neuroscience and neuroimaging communities.