Washington University

NIH Research Projects · FY 2024 · 2021-09

Abstract River blindness (onchocerciasis) is a major neglected chronic tropical disease that has been selected by the WHO for elimination by 2030. Currently, international control programs attempt to interrupt transmission of infection with annual mass drug administration (MDA), over the 10-14 years lifetime of the adult worms, using the microfilaricidal drug ivermectin that kills the microfilariae (mf) of Onchocerca volvulus, the causative agent of onchocerciasis. However, MDA with ivermectin is confounded in Africa because it cannot be used in areas co- endemic for loiasis due to the risk of severe adverse events. The overall goal of our project is to develop a novel direct macrofilaricidal (lethal to adult worms directly) preclinical candidate drug as targeted therapy for onchocerciasis using a multidimensional interdisciplinary experimental approach. Our proposal is based on two main resources we have established: a) an extensive omics resource that allowed the identification of defined conserved and diversified essential molecular targets in adult filarial worms, and b) a multifaceted screening funnel that was used successfully to phenotypically screen in vitro a library of drugs approved for clinical use, resulting in the identification of drugs with macrofilaricidal activity. In silico prioritization interfaced with the experimental identification of drugs that were active against the adult stage of filarial nematodes resulted in a short list of inhibitors with known drug-indication and putative target indication pairs that form the basis for our rational optimization of new compounds with direct macrofilaricidal potential. Our proposed aims thus build on our significant progress starting with validating the canonical targets in filarial nematodes, further expanding the list of our five high priority hit scaffolds with similar compounds by using pharmacophore searches, followed by discovery and rational design of innovative adult filarial nematode- selective drugs. The newly synthesized compounds will be optimized for potency and selectivity against the parasites vs. the host as well as against the cognate filarial nematode targets using a systems biology approach. We will also confirm the mode of action for the most promising lead compounds which will also be assessed for in vivo efficacy in two filarial small animal models that we have developed to facilitate assessing drug candidates in vivo (Brugia pahangi/jirds and Onchocerca ochengi/jirds). The overall approach deploys iterative optimization of two chemical series of compounds, and by the end of the project we plan to have at least one macrofilaricidal drug candidate to move into late stage preclinical studies. Overall, this project will emphatically address a critical research and thus operational gap; the identification and development of novel potent and safe macrofilaricidal drugs. Our rational discovery approach has the potential of providing macrofilaricidal drugs that kill directly adult worms needed to achieve the elimination goals for the human onchocerciasis.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Brain function requires coordinated activation of specific networks engaged in systems that process information in localised and distributed manners. In order to develop such specific networks, the brain engages groups of neurons that fire together in ensembles that can be observed with calcium imaging. Patterns of spontaneous activity in the cerebral cortex are thought to enable the formation of circuits specialised for processing different types of sensory information. How the brain first switches on activity across areas is unknown. I propose to investigate exactly how and when in fetal life these patterns first occur in vivo, what regulates their development, and how they shape neural circuits and later brain function. A major barrier to addressing this question has been that patterns of activity such as patchwork-type activity in S1 and travelling waves in V1 are present at birth in rodents making it difficult to study this question in vivo as the brain apparently switches on before birth. To address this, I propose to apply modern scientific tools and technologies to an Australian marsupial mammal: the fat-tailed dunnart (S. crassicaudata; Dasyuridae), thereby developing a new approach for investigating brain development. Dunnarts are small (adults weigh ~15g), carnivorous animals whose pups (joeys) are born at an equivalent stage of development to embryonic day 10 in mouse or seven-week gestation in humans, and therefore most of their brain development occurs as they develop inside their mother's pouch. Despite this more primitive developmental phase, dunnarts have a six-layered cerebral cortex which is similar to a mouse but with advantageous exceptions such as a more advanced binocular visual system. Dunnarts are also able to solve complex configurable problems and learn quickly. To ensure feasibility of this project, I provide evidence that we can use targeted electroporation to introduce sensitive calcium indicators such as GCaMP6S into the cortex. In preliminary experiments we find that patchwork-type activity in S1 and traveling waves in V1 are evolutionarily conserved in dunnarts, motivating this new direction of my research to understand the development and function of these patterns of spontaneous activity. Having access to study the entire genesis and development of these patterns enables longitudinal studies that can link cells, circuits and behavior/function. The creation of longitudinal imaging capabilities bridging micro/meso/macro scales as well as awake behavior across the lifespan will be required in order to identify which neuronal cell types initiate spontaneous synchronous activity and whether these activity patterns are instructive in forming functionally-specific circuits. I will also explore how spontaneous activity in the cortex evolves throughout life as circuits begin to function to mediate sensory experience and behavioural reactions. I ensembles knowledge propose that by understanding the fundamental processes r equired to build of patterned activity in the brain and how these affect behavior, this work will advance our of the neural basis of mental experience.

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically heterogeneous sample of the US population. To accomplish this, the Healthy Brain and Child Development (HBCD) Consortium has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,200 mothers and infants enrolled at 27 sites across the United States (US). The HBCD Study will carry out a common research protocol under direction of the HBCD Consortium Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD Data Coordinating Center (HDCC). The overarching goal of the HBCD Study is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a varied cohort that is representative of the US population; 2) pregnant woman with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD Study will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state-of-the-art methods. The HBCD Study will inform public policy to improve the health and development of children across the nation.

NIH Research Projects · FY 2024 · 2021-09

Short gut syndrome (SGS) results from the treatment of multiple conditions in adults and children. In children, the mortality associated with SGS is roughly 25%, making it one of the most lethal conditions in infancy and childhood. Morbidity among survivors is high with another 25% of children requiring a small bowel transplant. The current 5-year patient survival following a small bowel transplant is still roughly 58%. Intestinal failure associated liver disease (IFALD) represents a spectrum of liver injury including steatosis, cholestasis, fibrosis, and cirrhosis. IFALD is the leading indication for intestinal and/or multivisceral transplantation in children with SGS. The incidence of IFALD is roughly 50% in pediatric patients who receive parenteral nutrition (PN). The pathogenesis of IFALD is unique because SGS patients are enterally starved, have no insulin resistance, and are not obese. Using a PN-independent murine model of small bowel resection (SBR), we demonstrate perturbed gut barrier function and significant alterations in intestinal lipid signaling, severe hepatitis, cholestasis, necrosis, and regenerative nodules. In one mouse, we confirmed the development of HCC. Accordingly, our overarching hypothesis is that IFALD reflects a proinflammatory milieu within the remnant bowel along with profound alterations in lipid signaling within both intestine and liver to initiate hepatic injury, fibrosis, and ultimate progression to advanced liver injury. For this project, we have developed a multiple-PI proposal embracing world class expertise in intestinal adaptation responses to massive SBR (Warner/Rubin), intestinal and hepatic lipid signaling (Davidson) and genomics and metabolomics (Ding). In the first Specific Aim, we will focus on the intestinal contribution to liver injury and fibrosis. First, genetically altered mice will undergo SBR to determine the effect of impaired intestinal chylomicron assembly, disrupted intestinal expression of a major transcription factor involved with lipid sensing and signaling, and perturbed expression of an enterocyte cytoplasmic protein involved with absorption of long chain fatty acids on liver injury. We will then delineate the effects of varied dietary fat on liver injury, intestinal permeability, and portal venous cytokine production. Finally, we will determine the most important intestinal site of toll-like receptor 4 (TLR4) activity in the pathogenesis of altered gut permeability and resection-associated liver injury. The next Specific Aim will focus on the hepatic component of injury, steatosis, and fibrosis after SBR. We will delineate a temporal profile of lipidomic and lipogenic gene expression within the liver at multiple time points after SBR. We will determine whether alteration of the omega-6 to omega-3 ratio as well as disrupted expression of a major regulator of lipid synthesis contributes to advanced liver injury. Finally, we will elucidate a genomic and metabolomic profile in the liver of evolving liver injury in mice as well as in human patients with end stage IFALD. These findings may provide novel mechanistic insight into the etiology of IFALD.

NIH Research Projects · FY 2025 · 2021-09

Overall Project Summary/Abstract Cellular senescence has been characterized as a state of irreversible cell-cycle arrest coupled with a secretory program that can profoundly impact the tissue microenvironment. Our current understanding of senescence is largely based on cell culture and model-based studies. Research on the relevant signaling pathways and mechanisms underlying cellular senescence across human tissues over time is lacking. Our ability to leverage recent advances in omics and molecular imaging technologies enables us to investigate the transcriptional changes and secretory features driving and/or associated with senescence at higher depths and resolution than ever before. Here, we propose to develop the Washington University Senescence Tissue Mapping Center (WU-SN-TMC) within the NIH Senescence Network (SenNet). Our WU-SN-TMC will develop cellular senescence atlases using 500 human samples from four essential tissue types: bone marrow, breast, colon, and liver. We will first optimize our omics and imaging technologies and platforms for capturing, detecting, characterizing, and visualizing senescent cells; develop computational tools and models for accurate identification of senescent cells and markers; construct breast, bone marrow, colon, and liver senescence atlases in spatial and temporal contexts; and assess the landscape and heterogeneity of senescence. With these initial atlases, we will further characterize, validate, and define cellular senescence phenotypes and biomarkers using perturbation methods and investigate the interactions between senescent cells and the senescence-associated microenvironment. Finally, we will work with other SenNet centers to build comprehensive, major organ/tissue senescence atlases by integrated and comparative studies of all SenNet data across tissue types, time, sex, age, and ancestry groups. As a member of the SenNet program, WU-SN- TMC will employ state-of-the-art omics and imaging technologies, including bulk proteogenomics, single cell sequencing, spatial transcriptomics, CODEX molecular imaging, 3D light sheet microscopy plus expansion technologies that are likely to mature over the funding period, such as single molecule sequencing, to generate high-resolution, multi-parameter biomarkers and maps of cellular senescence in the four tissue types selected. We have the established infrastructure and expertise to successfully conduct this work, including high quality biospecimen collection, omics and imaging data production, experimental confirmation and validation, and high throughput, standardized, and reproducible data analysis. In conclusion, we will work closely with other SenNet centers and the Consortium Organization and Data Coordination Center (CODCC), to generate comprehensive atlases across major human tissue types under various physiological conditions, including changes across the human lifespan.

NIH Research Projects · FY 2025 · 2021-09

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) has been providing valuable references for the research community studying urogenital development and diseases. It is a recurring theme that rapidly advancing new technologies are instrumental in enhancing and expanding reference databases. Cross fertilization of atlas building efforts spanning various organ systems and disease types will undoubtedly boost the technology penetration across these projects. To build multi-dimensional atlases of developing urogenital organs that incorporate the latest multi-omics and spatial molecular mapping technologies, we have assembled a team with expertise both in urogenital development and multi-dimensional, multi-platform, molecular atlas building. We propose to utilize the infrastructure we developed at our institution for the NCI Human Tumor Atlas Network (HTAN) and other large scale projects as a springboard to help effectively and efficiently propel GUDMAP to the next level with transcriptome-wide coverage, single cell level resolution, and spatial mapping with unprecedented clarity. We will take advantage of our experience in the incorporation of single nucleus (sn) RNA-seq and snATAC-seq to establish a comprehensive epigenetic and transcriptomic landscape in targeted urogenital organs and structures (lower urinary tract (LUT), selected male reproductive organs, kidney vasculature, lymphatics, and nerves) at single cell resolution (Aim 1). We will then add the spatial dimension to this molecular landscape to build 2D and 3D molecular atlases by incorporating spatial transcriptomics (ST), CODEX, and light sheet microscopy (LSM) (Aim 2). In Aim 3, we will extend our study to disease models, focusing on murine models of congenital anomalies of the kidney and the urinary tract (CAKUT). With the proposed experiments, we aim at building multidimensional molecular atlases for developing urogenital organs at unprecedented cellular resolution and gene coverage with the highest efficiency possible. Aim 1: Characterize the epigenetic and transcriptomic landscapes of developing urogenital organs with single cell omics We will perform integrated transcriptomic and epigenetic profiling of the developing/maturing LUT, male reproductive organs, and the kidney at E16.5, NB, and 3 weeks of age. Although the focus for the kidney will be on vasculature, lymphatics, and nerves, since there is a lack of single cell omics data on most of the selected stages, our data will also help to strengthen GUDMAP data for the broadest use by the research community. Aim 2: Construct multi-dimensional molecular atlases for developing urogenital organs using spatial transcriptomics and advanced imaging technologies A major challenge for atlas building in biological systems has been spatially assigning large number of molecular features to the anatomical and cellular structures. We have successfully established experimental procedures and computational analyses pipelines for spatial transcriptomics, CODEX, and light sheet microscopic imaging, to map gene expression data, including transcriptome-wide data to cells and structures. We will use these technologies to analyze the developing/maturing lower urinary tract, male reproductive organs, and the kidney at E16.5, NB, and 3 weeks of age for the construction of truly multi-dimensional, multiplatform molecular atlases. Aim 3: Building molecular atlases for key urogenital structures using murine CAKUT models with cell ablation or gene inactivation CAKUT occurs in many different forms representing a significant cause of morbidity and mortality in the pediatric population. We have generated and analyzed several CAKUT murine models in the past. Building atlases of the target organs for these models will provide high resolution, spatially registered molecular references for key stages of disease initiation and progression. Moreover, such atlases will help researchers better understand normal urogenital development by knowing the tolerance of the systems and processes in dealing with various disturbances. We will use a highly reproducible murine model of CAKUT with inactivation of canonical Smad signaling in ureteral mesenchyme, causing a uniform ureteropelvic junction (UPJ) obstruction phenotype prenatally. We will use the technologies outlined in Aims 1 and 2 to build molecular atlases of the relevant structures (ureter, kidney, UPJ) at key time points and compare the atlases of defective development with those of normal development.

NIH Research Projects · FY 2025 · 2021-09

Abstract Alzheimer's disease (AD) is a highly heterogeneous multifactorial disease. Genetic influences on AD are strong as shown by several pathogenic genes and over 50 AD loci identified through genome-wide association studies (GWAS). There are also clear sex differences in AD risk and progression. Women are at a higher risk of developing AD and present faster progression. A recent GTEx study also highlights sex differences in the genetic regulation of gene expression. Despite these established sex differences, sex-specific molecular findings in AD are still limited. The objective of this study is therefore to generate detailed sex-specific multi- tissue molecular profiles of AD and decipher the genetic architecture that underlies AD. We propose to identify sex-specific functional mechanisms underlying the genetic architecture of AD. We will generate multiple layers of -omics data, including DNA methylation, gene expression, proteomics, metabolomics, and lipidomics, from several large and well characterized studies. A series of well-powered sex-specific omics characterization across multiple tissues can help identify novel drug targets and provide critical insights for clinically translatable interventions for prevention and treatment. We will then map additional GWAS loci by performing sex-specific multi-omic quantitative trait loci and co-localization for each -omic layers. We will identify the causal genes, proteins, and additional -omic analytes by performing Mendelian randomization. Analyzing such omics data will elucidate a causal path from sex-specific genetic variation to AD risk, onset and progression. The human multi- omic data will finally be combined with induced pluripotent stem cell (iPSC) models to identify novel sex- specific FDA-approved therapeutics. We have assembled a very productive and interdisciplinary research team with expertise in all the aspect of the proposal. All aims in this proposal will be conducted and reported in compliance with NIH guidance on scientific rigor and reproducibility. Our preliminary data already identified several genes and proteins that are associated with AD risk and cerebrospinal fluid biomarkers in a sex- specific manner. All the raw data will be shared via NIA-approved mechanism (including AD Knowledge Portal, NIAGADS). This rich resource will benefit the field for additional analyses beyond the ones proposed here.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The landmark Healthy Brain and Child Development (HBCD) study will provide a representative reference data resource to the scientific community enabling unprecedented investigation of neurodevelopment and the impact of environmental, genetic, and biological factors on brain and behavioral health and developmental trajectories from infancy through childhood. Through this study, the Healthy Brain and Child Development Consortium will recruit and retain a cohort of approximately 7,200 pregnant women from 24 sites across the U.S. and follow these families and their children through the first decade of life. Children will undergo rigorous data collection across modalities including neuroimaging, neurophysiology, behavioral and cognitive assessments, and collection of biospecimens via a balanced protocol developed by field-leading experts. Building upon the substantive complementary experience and expertise of its multidisciplinary team and leveraging multiple population-specific technical innovations, the Healthy Brain and Child Development Data Coordinating Center (HDCC) will provide the leadership, management, and oversight of data collection, quality control, curation, processing, management, sharing, and analytics to facilitate and support the activities of the HBCD Consortium and ensure its success. Included is development and implementation of an optimized, state-of-the-art MRI protocol harmonized for the first time in infants/toddlers across all three major vendors which leverages the latest innovations in scanner technology with age-specific structural, microstructural, quantitative, functional, and spectroscopy sequences. Also detailed is a targeted EEG protocol linked with a field-leading automated processing pipeline for developmental EEG which provides innovative derivative measures. Data and project management will occur through a centralized tracking and distribution platform linked to a high-throughput compute backbone which overcomes limits of commercially-available systems for management and integrated processing of multimodality data from large, multi-site studies. High performance computing will be supported through unique access to a combination of field-leading resources. Detailed procedures are outlined for secure collection, management, and analysis of personally identifiable information (PII) data, including flexible methods designed to accommodate heterogeneity in electronic health record systems across sites. Finally, substantive HBCD-specific enhancements to the Data Exploration and Analysis Portal (DEAP 2.0) will produce a crucial tool for data access to authenticated users while promoting best practices in reproducible statistical analysis and providing flexible computation without the need to download restricted-access data. The result of this field- leading combination of HDCC resources will be a state-of-the-art, longitudinal data set of unparalleled scale which provides deep understanding of the biological and environmental factors that affect a child’s health, brain, and behavioral development and shapes research, clinical care, and public policy for decades to come.

NIH Research Projects · FY 2026 · 2021-09

ABSTRACT FOR OVERVIEW The Aging Adult Brain Connectome (AABC) leverages the existing infrastructure developed by the Human Connectome Project for Aging (HCP-A) by obtaining longitudinal follow-up data (neuroimaging, cognitive testing, and blood) using a standardized protocol from a well characterized cohort of over 1,000 healthy individuals to generate within-participant brain trajectories for up to 10 years. At initial recruitment, individuals enrolled in the HCP-A were generally physically and cognitively healthy but over time some will develop preclinical AD or early cognitive changes due to AD or ADRD. The AABC is comprised of four Projects: Project 1 examine the effects of stress and allostatic load, including inflammation, during the early adult period. Project 2 examines the effects of lifestyle behaviors on the trajectory of cognitive and brain changes during the mid adult period. Project 3 examine the effects of menopause transition/vasomotor symptoms during the mid adult period. Project 4 exam- ine the clinical and neural indicators of resiliency and resistance to AD and ADRD in the later decades of adult- hood. The AABC also consists of 4 Cores: The Administration Core (AC) will provide essential core and site leadership to carry out the scientific mission of the AABC. The diversity recruitment and retention unit (DRRU) will be located within this core and will ensure that the AABC continues to recruit and retain an adequate distri- bution of races that is currently seen in the US. The Integrated Data Acquisition Core (IDAC) provides expertise and personnel from each site to acquire high quality neuroimaging, deep phenotyping of non-imaging data, and biosamples from each site.The Informatics, Data Analysis, and Statistics Core (IDASC) will house project imag- ing data using the IntraDB database, will perform quality control of raw and analyzed data, will develop and run cross-sectional and longitudinal pipelines to produce multi-modal imaging data phenotypes for each project, will provide dimension-reduced summaries, will impute missing data; and will develop and run statistical models for each project. The IDASC will also be responsible for data sharing with the general public. The Genetics and Multi-omics Specimens Core (GMSC) will provide genetic information on participants evaluated through the AABC who have been characterized using a uniform protocol. Multi-omic data and AD biomarker data will be generated by the GMSC.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Protein-losing enteropathy (PLE) is the term given to the pathological phenomenon of protein dumping from the systemic circulation into the intestinal lumen. Various degrees of PLE are observed in a variety of otherwise unrelated diseases. For instance, PLE is a complication of the life-saving Fontan palliation procedure performed on children born with only a single ventricle of the heart to create a vascular diversion to the lungs to promote improved oxygenation of blood. Fontan-associated PLE is linked with high mortality: 30-50% over 5- to 10-years. Yet, PLE remains understudied. The dominant causal mechanisms underlying PLE appear to relate to (1) loss of barrier integrity at the intestinal epithelium or (2) functional disturbance of the lymphatic vasculature. Intestinal epithelial cell erosion would expose proteinaceous tissue fluid to the intestinal lumen. Furthermore, poor epithelial intercellular junctions might allow for the contents of the lamina propria interstitium to leak into the intestinal lumen. With respect to lymphatics, failure of lymph to flow away from the intestine uni-directionally toward the heart and instead to flow backwards from the body trunk toward the intestine with sufficient force to break across the epithelium may be a major cause of PLE. The literature presents Fontan palliation-associated PLE as a problem driven by lymphatic backflow, whereas PLE in IBD (such as Crohn's disease) is presented as being of mixed etiology that involves breach of epithelial integrity with possible additional contributions stemming from lymphatic dysfunction. However, it is acknowledged in the literature that the full basis of PLE in any of these conditions is uncertain and that there are several reasons why the current explanations may be questioned. We will carry out focused research on PLE that includes studies involving human participants as well as studies in experimental animal models. Our hypotheses are, first, that the lymphatic vasculature is a primary player in PLE affecting Fontan patients and IBD patients, and second, that the lymphatic vasculature must receive “two hits” to drive sufficient backflow of lymph to cause outflow from the intestinal barrier. The first of these hits has already been considered (but not completely tested) in the context of Fontan palliation: increased pressure within the chain of lymphangions (vessel units between lymphatic valves). We propose this second hit is an inflammatory signal that negatively affects lymphatic valves. The two-hit model may resolve a confusing observation in Fontan patients with PLE wherein treatments with steroids like budesonide can be effective. A steroid seems unlikely to strongly alter pressure in the venous and lymphatic systems, but it is easy to envision how steroids may help by reducing adverse inflammatory signalling associated with valve failure. If this model is correct, a path to therapies not previously considered to treat PLE may become evident.

NIH Research Projects · FY 2025 · 2021-09

Abstract Infectious osteomyelitis (OM) is an inflammation-driven disease of bone that culminates in pathological alterations in skeletal architecture. Bone infections are multifactorial and reflect a complex interaction between microorganisms and host cells. Staphylococcus (S.) aureus, a pathogen that has developed antibiotic resistance, is the leading cause of bacterial-induced OM and has been identified as one of the greatest bacterial threats to global public health. These infections are painful, debilitating and can become chronic or recur years after the initial event. The pathogen’s ability to damage bone tissue and evade clearance by the immune system, even with appropriate antibiotics, impose significant obstacles to treatment of OM. The first and most critical level of host defense against infection by S. aureus is innate immunity, primarily mature myeloid lineage cells such as neutrophils and macrophages; the success of this pathogen is dependent on its ability to evade and exploit these responses. While much has been learned about interactions between myeloid cells and S. aureus, relatively little work has specifically focused on infections of bone. This microenvironment presents unique features, including relative hypoxia, abundant immature myeloid cells, and the presence of unique bone cells – osteoclasts (OCs), osteoblasts, and osteocytes - that interact with both the bacteria and innate immune cells. Furthermore, the route of infection - via injury or direct soft tissue extension, surgical implants, or hematogenously spread – can significantly alter the interactions between bacteria and bone, especially during early stages of infection. Notably, OCs differentiate from monocytic precursors, providing an inherent link between immature myeloid lineage cells and bone homeostasis. The overall goal of this application is to understand the host-pathogen interactions between the bone’s OC and neutrophil lineage cells and S. aureus during the establishment, progression, and resolution of OM. Our preliminary studies strongly implicate the interleukin-1 (IL-1) signaling axis as a driver of both antibacterial immunity and pathologic bone changes during OM. Following infections such as with S. aureus, IL-1 family members including IL-1β are canonically generated through the activation of multi-protein complexes known as inflammasomes. However, little is known about the role of inflammasomes in the pathogenesis of OM. We have found that, compared to their uncommitted precursors, OCs have lower inflammasome activation and are permissive of intracellular S. aureus proliferation. We hypothesize that differences in inflammasome activity within myeloid lineage cells present in bone affect the pathogenesis of OM, with S. aureus exploiting those cells with weaker inflammasome and antimicrobial responses as a proliferative niche while leading host cells with an excessive inflammatory response to cause tissue damage. Aim 1: Define host and pathogen determinants of inflammasome activation in the OC lineage in OM. Aim 2: Define the mechanisms and impact of inflammasome activation in the neutrophil lineage by S. aureus in OM. By examining and manipulating the host-pathogen interactions in specific myeloid cell populations, we will learn how to tip the balance towards resolution of OM.

NIH Research Projects · FY 2024 · 2021-09

Project Summary/Abstract: There is an urgent need to develop new approaches to induce broadly protective immunity against rapidly mutating viruses. The induction of protective immunity requires the development of memory lymphocytes that can rapidly recognize and neutralize the virus upon re-infection. Virus-specific memory B cells are long-lived cells that are critical for the establishment of protective immunity by vaccines. Memory B cells mediate protective immunity by rapidly differentiating into antibody secreting cells upon antigen re-encounter. Memory B cells that re-encounter antigen can also undergo further mutation of their B cell receptor to increase their affinity for viral antigen. Induction of broadly reactive memory B cells through iterative exposure to cross- reactive viral antigens is an emerging vaccination strategy designed to protect against rapidly mutating viruses. However, efforts to induce broadly reactive memory B cells have been hindered by the relative inefficiency with which memory B cells diversify their B cell receptor following antigen re-encounter. Better understanding of the mechanisms governing memory B cell development is necessary to facilitate the design of vaccines capable of eliciting a broadly protective memory B cell response. Additionally, effective harnessing of memory B cells to protect against mucosal viruses requires understanding how memory B cells develop and function in barrier tissues. Memory cells are critical for the establishment of barrier immunity, with lung-resident memory B cells recently identified as essential to elicit a rapid and robust local antibody response following influenza challenge. However, a lack of clarity regarding the pathways regulating memory B cells development in barrier tissues remains a significant barrier to designing vaccines capable of inducing tissue-resident memory B cells. This project proposes to characterize how the tissue microenvironment shapes the development of memory B cells following viral infection. This project will first determine how viral- and tissue-specific cues influence the transcriptional profile of memory B cells. This proposal will then identify key transcriptional regulators of memory B cell development in barrier tissues and investigate the mechanisms by which these regulators function. Transcriptional regulators are essential in facilitating memory lymphocyte development and function in barrier tissues. By revealing the developmental pathway of memory B cells in barrier tissues, this study will provide important insight into how vaccines can overcome existing challenges in order to elicit broadly protective immunity against mucosal viruses. The development of vaccines specifically designed to induce tissue-resident memory B cells would mark an important paradigm shift away from traditional vaccination approaches focused on inducing a systemic virus-specific B cell response.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Kidney disease is worldwide health problem that is becoming increasingly common. Primary glomerular disease, both genetic and acquired, represents a significant proportion of cases. We are interested in understanding the makeup of the glomerular filtration barrier and how it becomes damaged, leaky to plasma proteins, and eventually non-functional. Our focus has been to investigate the composition and function of the glomerular basement membrane (GBM), a specialized extracellular matrix that is an integral component of the kidney’s filtration barrier. The GBM contains collagen IV, laminin, nidogen, and the heparan sulfate proteoglycan agrin, and likely dozens of other less abundant matrix proteins. Although the GBM is synthesized by both podocytes and glomerular endothelial cells, it is exclusively podocytes that make the major collagen IV isoform, which consists of the α3, α4, and α5 chains that assemble to form a secreted heterotrimer. Mutations that affect this collagen IV component of the GBM cause Alport syndrome, which leads to end-stage kidney disease (ESKD) as well as hearing and eye defects. The prevalence of Alport syndrome has been estimated to be 1 in 5,000 to 10,000 newborns, so there are hundreds of thousands of affected patients around the world. Structural GBM abnormalities secondary to the collagen IV defect lead to thickening and splitting of the GBM and eventually podocyte foot process effacement, glomerulosclerosis, and tubulointerstitial fibrosis. Until recently there has been no treatment for Alport syndrome. However, studies in mice and dogs had shown that ACE inhibition slows kidney disease progression to ESKD. These animal studies have been validated in human Alport syndrome patients, for whom ACE inhibitors or angiotensin II receptor blockers are now considered the standard of care. Despite this treatment breakthrough that delays ESKD, it is not a cure; there is still a need for new targeted therapies. The goal of this proposal is to use innovative, state of the art technologies that involve small molecule, genetic, and protein biochemistry approaches to attempt to either restore the Alport GBM to a somewhat normal composition or to alter its composition through removal of pathogenic components. In either case, the expectation is that improving GBM composition will at least partially normalize GBM structure and function. Together with renin-angiotensin system blockade as a standard of care, these treatments should greatly delay the time to ESKD and be beneficial for patients.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The prognosis for pancreatic ductal adenocarcinomas (PDAC) patients is dismal. This is likely due to the presence of a uniquely suppressive tumor microenvironment (TME) that is dominant in most PDAC. Our data suggest immune priming by conventional dendritic cells (cDCs) may be a necessary barrier to overcome to generate lasting immunity in PDAC patients. cDCs are central for generating tumor antigen–specific T cell responses. Our new data show that cDCs are severely dysfunctional in patients with PDAC. This dysfunction is driven by two mechanisms: 1) We recently reported that PDAC patients have impaired cDC development in their bone marrow, and this leads to functional depletion of circulation pre-DCs, and poor response to checkpoint inhibitors. 2) We recently showed that even when cDC development is not fully impaired, cDC1s are physically/biochemically excluded from the PDAC TME. These mechanisms to the loss of stereotactic body radiation therapy (SBRT)-induced priming of tumor antigen-specific T cell responses and ultimately failed tumor control in animal models. We overcame both of these dysfunctional barriers by targeting cDC1s using a combination of systemic treatment with FMS-like tyrosine kinase 3 ligand (FLT3L) and CD40 agonists. Our pre- clinical data are exceptionally strong and have placed us in a unique position to translate these findings into PDAC patients. Our central hypothesis is that targeting cDC can unlock responsiveness to RT by generating lasting anti-tumor immunity. We will expand test this hypothesis in three specific aims. Aim 1. Determine the safety and efficacy of the combination of CDX-301 plus CDX-1140 and SBRT in locally advanced PDAC patients Aim 2. Determine the mechanisms by which FLT3L plus a CD40 agonist induce anti-tumor immunity. Aim 3. Determine if FLT3L plus CD40 agonists improves responsiveness to checkpoint immunotherapy. Impact. PDAC patient responses to conventional radiation therapy have been disappointing. Our data strongly support the use of FLT3L and CD40 agonist to enhance patient responsiveness to RT and generate long-term anti-tumor immunity. Our team is well-positioned to test our central hypothesis directly in clinical and experimental studies.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT U2R Research Training Program (RTP) grant application for DS-I Africa initiative (RFA-RM-20-016) is a partnership of 3 collaborating institutions: Washington University in St. Louis (WUSTL), University of Rwanda (UR) and African Institute for Mathematical Sciences (AIMS), both in Kigali, Rwanda. The overarching objective of U2R RTP is to stimulate and catalyze innovation of Data Science (DS) for Health in Rwanda. A high prevalence of communicable diseases, coupled with a rapidly expanding epidemic of non-communicable diseases (NCDs), forecasts a perfect public health storm in Rwanda, providing impetus and rationale to leverage DS to address health care gaps. A structured program design will help develop trainee research careers in DS with particular focus on health care topics relevant to Rwanda, including communicable (i.e., HIV, malaria, COVID-19, etc) and chronic NCDs (i.e., hypertension, heart disease, diabetes, etc). We propose to expand existing model curriculum for innovative and interdisciplinary DS for Health research training and practice by identifying additional competencies in three major scientific areas: 1) computer science & informatics; 2) statistics & mathematics; and 3) biomedical sciences & public health. U2R RTP will: a) provide support (tuition, stipend, research funds) for 24 trainees: MS (n=14), PhD (n=6); post-doc (n=2); junior faculty (n=2); each trainee receives training for 1-2 years based on individual needs; b) foster an innovative team science transdisciplinary approach to research; and c) build institutional capacity at UR and AIMS to support the long- term sustainability of the program. The intended outcomes are to: 1) train next generation of data scientists that will have the necessary expertise to solve challenging problems presented by the burden of communicable and chronic NCDs in Rwanda; and 2) complete a full U2R program leadership and faculty and institutional transition from US-Rwanda co-led to fully Rwandan-led by the end of the U2R program. Trainees will participate in mandatory week long “boot camps” (twice/yr), monthly webinars and mid-yr mtg with didactic training focused on career advice, research and academic advancement in highly-relevant Data Science and health care topics (i.e., data management, bioinformatics, statistics, epidemiology, communicable diseases, NCDs, environmental exposure, dissemination and implementation, etc); intense Grant & Manuscript Writing Skills Training Program geared towards development of competitive grant proposals (“Small Research Projects, SRP)” that trainees prepare, submit and conduct under direction of a multidisciplinary mentoring team from academic and governmental institutions. Effective mentoring during boot camp occurs through trainee “Group Brainstorming Sessions” where they present their developing SRPs to obtain feedback. An extensive program evaluation by trainees and faculty will also provide feedback for improvement. The U2R RTP will establish and cultivate a sustainable program for training, research, mentoring and career development that will position UR and AIMS as national and regional African leaders and a global hub for Data Science for Health in Rwanda.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Triple negative breast cancer (TNBC) is aggressive and a large percentage of patients develop metastatic disease. Disseminated tumor cells (DTCs) found in the bone marrow (BM) of TNBC patients may be the intermediaries of the metastatic process. Data from our lab as well as others suggest that the immune landscape of BM may influence DTC latency, treatment resistance, and metastatic potential. We have already defined and validated an 8 gene expression-based biomarker panel that can detect DTCs in the BM of treatment naïve TNBC patients and that predicts development of distant metastatic disease. Our recent data indicate that TNBC patients with DTC-positive BM have altered populations of immune cell precursors and this is associated with recurrent disease development. Based on these findings, we hypothesize that immune checkpoint inhibitors will facilitate the elimination of BM DTCs in TNBC patients by altering the immune microenvironment in patients with specific DTC and/or BM immune cell populations, and that cell population-specific gene expression signatures can predict which patients will benefit most from aggressive immunotherapy to prevent metastatic disease relapse. We will test this hypothesis using our extensive biorepository of BM specimens collected from TNBC patients who received conventional chemotherapy, as well as prospectively collected specimens from TNBC patients participating in an independently funded institutional phase II immune checkpoint inhibitor (ICI) trial of carboplatin/paclitaxel/nivolumab with or without cabiralizumab. Our goals are: 1. To evaluate the ability of our 8- gene DTC gene panel to predict distant disease development in TNBC patients enrolled in our ICI therapeutic trial of carboplatin/paclitaxel/nivolumab with or without cabiralizumab; 2. To understand the specific subpopulations of BM DTCs in TNBC patients treated with conventional chemotherapy and ICI therapy which are resistant to therapy, and; 3. To understand alterations in specific T cell and conventional dendritic cell (cDC) populations in the BM when DTCs are present, and how this is impacted by conventional and ICI therapy. The results of this proposal will lead to a greater understanding of immune escape and heterogeneity of BM micrometastatic disease as well as biomarkers for improving conventional and ICI therapy in TNBC patients.

NIH Research Projects · FY 2024 · 2021-09

Project Summary This project is to reveal the mechanism of how voltage, phosphatidylinositol 4,5-bisphosphate (PIP2) and calmodulin (CaM) integrate to activate the IKs potassium ion channels in the heart. The IKs channel is important for repolarization of cardiac action potentials and the control of heart rhythm. CaM and the membrane lipid PIP2 are important cell signals, and meanwhile these molecules are cofactors of the IKs channel required for channel opening. These enable the IKs channel to play a critical role in the adaptation of heart rhythm to various physiological conditions. Congenital and drug induced IKs malfunction is associated with cardiac arrhythmias. Previous studies and our preliminary data indicate that the IKs channel is a novel target for antiarrhythmic therapy. The significance of this study is to improve the understanding of molecular basis of cardiac electrophysiology and antiarrhythmic drug development. At present how CaM and PIP2 interact with the IKs channel is not clear. This proposal is motivated by the recently published structural data of the channel protein. Combining these data with other published and our preliminary studies lead to an exciting hypothesis for how CaM, PIP2 and voltage integrate to activate the IKs channels. Our specific aims are designed to examine three key aspects of this hypothesis. We will use electrophysiological approaches, fluorescence spectroscopy, structure-informed mutagenesis and molecular dynamic simulations to study the IKs channels expressed in exogenous expression cells including Xenopus oocytes and mammalian cell lines. These studies will reveal the binding of PIP2 and CaM to the channel protein and subsequent conformational changes that open the channel. These results will provide a molecular basis to understand how heart rhythm is regulated by cell signals and multiple mechanisms for drugs to target and modify, which may lead to the development of more effective and safe antiarrhythmic drugs.

NIH Research Projects · FY 2025 · 2021-09

Multiple myeloma (MM) is a common and lethal hematologic malignancy. Treatments of MM have been rapidly evolving. While these new treatments improve survival considerably, the median survival still ranges from 43-83 months at diagnosis. Among all cancer sites, the management of MM is the costliest, which in part can be attributable to guideline recommended multidrug regimens. Despite such significant health and economic burdens and rapid changing landscape for MM treatments, MM is not one of the cancer sites in the Cancer Intervention and Surveillance Modeling Network (CISNET). Therefore, MM lacks comparative modeling to set goals and policy prioritization in MM prevention and control. Moreover, unlike breast cancer or colorectal cancer, there exists no population-based screening for MM or risk managed strategies for those with premalignant conditions (MGUS and smoldering MM). MM requires comparative modeling to evaluate promising intervention strategies, particularly at premalignant stages. To prevent/control this devastating disease, it is imperative to demonstrate the potential benefits and harms of the novel interventions before implementation. It is also important to demonstrate the potential benefits and harms of de-implementation of the existing interventions, in lieu of the novel ones. This Incubator Program will include two modeling groups to conduct comparative modeling under the coordination of the coordinating center. Our Program will evaluate novel strategies in preventing or treating MM with the goal of reducing the health and economic burden of MM. We plan to comparatively build, calibrate, and validate evidence-based MM modeling across the MM care continuum (Aim 1). Using the proposed comparatively modeling, we will (1) assess the impacts of novel MM prevention strategies in high-risk patients diagnosed with MGUS (Aim 2); (2) evaluate the cost-effectiveness of novel treatment regimens as well as guideline-recommended treatments in patients diagnosed with MM (Aim 3); and (3) assess whether, under what conditions, and in which ways the goal of minimizing health and/or economic burden can be achieved through the proposed novel intervention strategies (Aim 4). The proposed MM Incubator Program is significant in its capability to 1) build evidence-based comparative modeling for MM, a disease area that lacks of such modeling, relative to the areas of solid tumors already with such modeling, to guide interventions and policies; 2) provide evidence-based evaluation before implementation of any costly clinical trial or de-implementation of any outdated intervention; 3) explore novel interventions/treatments at various stages of MM; and 4) examine the value of guideline-recommended therapies, providing evidence to inform changes in guidelines and thus a shift in current clinical practice of MGUS and MM management. The proposed intervention strategies for MGUS and MM patients are innovative, with the goals to prevent and control MM. Successful completion of this study will provide evidence in tangible metrics to urge a paradigm shift from current MGUS/MM management. It is therefore a vital step to move the field forward.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT The host inflammatory response is a double-edged sword that must vigorously defend against pathogens, but also requires restraint to prevent unintended injury to the host. The Cytokine Storm Syndrome (CSS) represents a state of unbridled inflammation that can be triggered by infections, including Severe Acute Respiratory Syndrome-associated Coronavirus 2 (SARS-CoV-2). While evidence for dysregulated cytokine responses exists for the SARS-CoV-2-associated CSS (S-CSS), the precise cell types and viral factors that precipitate this response remain incompletely understood. Autophagy is a cytosol-to-lysosome degradative pathway that has important functions in host immunity. We have recently shown that autophagy genes in myeloid cells, preliminarily alveolar macrophages (aMΦ), confer protection in a murine model of CSS induced by intravenous TNF. We hypothesize that host autophagy may also have a pleiotropic role in limiting the S-CSS. We are motivated in this hypothesis since coronaviruses (CoVs) manipulate host autophagy-associated membranes for their own replication via the nonstructural protein 6 (nsp6). This proposal for the NIH Director's New Innovator Award will test the role of host autophagy and a viral antagonist in the triggering of S-CSS using both established and innovative methods. The project will utilize a model for SARS-CoV-2 infection in which the human ACE2 receptor (encoded by hAce2) is delivered to mouse lungs via adenovirus (AdV) vector. Additionally, we will determine the role of aMΦ-specific host pathways by utilizing mice deficient for GM-CSF signaling and devoid of aMΦ (Csf2rb-/-), that are durably restored with aMΦ by a single intranasal instillation of progenitor cells in neonates. The role of SARS-CoV-2 nsp6 in viral pathogenesis will be determined with recombinant viruses deleted for this factor or with naturally occurring point mutations hypothesized to facilitate infection. Moreover, we will develop an AdV-hAce2 vector system expressing sgRNAs to edit genes directly in susceptible respiratory cells in Cas9-transgenic recipient mice. We will generate pooled AdV sgRNA libraries via this method for in vivo screening approaches that may identify host pathways important for regulating infection not otherwise recapitulated by in vitro approaches. Further, we will reconstitute Csf2rb-/- mice with aMΦ cell progenitors containing pooled CRISPR libraries to identify host genes important for not only the aMΦ response to SARS- CoV-2 but also for fundamental aspects of aMΦ niche development. These studies have the potential to identify new areas for the development of host- and viral-directed therapies (e.g., the autophagy pathway and nsp6, respectively). The robust and versatile in vivo platforms established for functional genomic studies of a tissue site critical for the proximal response to SARS-CoV-2 have broader implications for the study of complex cell populations in diverse biological processes.

NIH Research Projects · FY 2025 · 2021-09

Summary/Abstract We have limited understanding of how uterine contractions develop and become sufficiently coordinated to expel the fetus at term. For example, we lack answers to basic questions about labor such as where contractions initiate, how fast contractions propagate, which regions of the uterus are active during contractions, and how these measures change as labor progresses. To address this knowledge gap, a new imaging technology, Electromyometrial Imaging (EMMI) was recently developed and validated in a translational sheep model. EMMI employs magnetic resonance imaging to acquire a subject-specific body-uterus geometry, then combines the resulting data with electrophysiological data collected from up to 256 electrodes on the abdominal surface. With EMMI, it is possible to noninvasively image the electrical activation and conduction patterns during contractions across the entire uterus in three dimensions. Preliminary data indicate that EMMI can be used to systematically characterize contractions during normal term labor in humans. Additionally, three proposed features of uterine electrical activity, termed "contraction indices", appear to correlate with time until delivery. The objectives of this proposal are to use EMMI to create a "normal term atlas" describing the 3D electrical activation patterns of human uterine contractions at high spatial and temporal resolution across labor, and to use this atlas to begin to identify contraction features associated with impending labor arrest. Aim 1 is to define the uterine electrical maturation and contraction patterns during labor in term nulliparous women. In this Aim, EMMI will be conducted on 430 nulliparous women throughout labor, and data will be analyzed from the 365 women who are anticipated to have normal term labor. This aim tests the hypothesis that at least one of the EMMI-derived uterine contraction indices can precisely reflect progression of normal term labor in nulliparous women, and can reliably differentiate the different phases of the first stage of labor. Exploratory Aim 2 will evaluate uterine electrical contraction patterns during labor in the anticipated 75 women from Aim1 who experience labor arrest. This exploratory aim will provide the basis for a future larger EMMI study to fully characterize the spatial-temporal signatures of uterine contractions in patients who develop arrested labor. In completing these two aims, this project will generate physiologically normal standards of uterine contraction indices of nulliparous women during the progress of term labor. These normal standards will permit future in-depth clinical investigations of the factors leading to dysfunctional labor. Moreover, they may, in the longer term, serve as standards for monitoring pregnancy and labor progression and assessing the effectiveness of treatment strategies to manage labor and prevent labor complications such as preterm birth, labor arrest, and postpartum hemorrhage.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Problematic substance use is associated with significant personal and socioeconomic costs (accounting for approximately 5% of global disease burden and worldwide deaths). Substance use initiation, progression to heavy use, and early onset substance use disorders (SUDs) commonly emerge during adolescence and young adulthood. This developmental period of risk is theorized to result from typical patterns of regionally asynchronous brain maturation (i.e., rapid and early development of limbic regions alongside relatively immature prefrontal and multimodal association cortices) resulting in a diminished ability to suppress inappropriate emotions, desires, and actions when salient environmental cues are present. During later young adulthood the stabilization, reduction, or desistance of heavy use typically occurs alongside maturing cognitive control and emotional regulation abilities coinciding with cortical development. Brain and behavioral maturation may also be influenced by substance use. As genetic and environmental risk factors for substance involvement are predominantly shared across substances, understanding the behavioral and neural mechanisms underlying these shared risk factors in a developmental context will broadly improve our etiologic understanding of substance involvement liability and refine treatment and prevention. In this 5-year R01 (responding to PAR-19- 162), we propose to test whether putative behavioral and neural mechanisms of stage-based addiction may link broad spectrum SUD genomic liability and environmental risk to substance involvement trajectories from childhood – young adulthood using longitudinal data from the Adolescent Brain and Cognitive Development (ABCD) Study (N=11,875 followed from ages 9-16) along with other samples that uniquely extend the temporal scope of ABCD to comprehensively examine brain-behavior developmental interplay related to substance use and misuse (e.g., National Consortium on Alcohol and Neurodevelopment in Adolescence followed 830 individuals from ages 12-32). Disentangling the behavioral and neural mechanisms underlying broad spectrum genetic and environmental liability to SUD will inform our etiologic understanding of substance use initiation, escalation, and desistence that may ultimately contribute to substance-related policy, education, nosology, prevention, and treatment. Primary deliverables from this project will be manuscripts evaluating whether behavior and neural phenotypes may represent mechanisms underlying polygenic and polyenvironmental risk for substance use disorders.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract Skin disease affects 1 in 4 Americans and costs $75 billion annually. Diagnosis relies upon visual inspection, but the subjective nature of visual assessment can lead to death from skin cancer misdiagnosis or suffering from misdiagnosis of a treatable chronic inflammatory condition. Non-white Americans make up 40% of the population and face further challenges, because disease can appear more subtly in pigmented skin, leading to systematic underdiagnosis and undertreatment. Skin biopsy is more objective but not all patients, particularly children, tolerate invasive procedures, and there are concerns about overutilization and low diagnostic yield of biopsies. Thus there is an urgent need for objective technologies that can quantitatively assess cutaneous inflammation and neoplasms across diverse skin types, and that can provide noninvasive alternatives to skin biopsy. Macroscopic optical imaging modalities may be able to address this need, but mostly depend on Silicon(Si)-based photodetectors, which constrains what these devices can measure because in the range where Si-based detectors are most sensitive (400-950 nm), melanin and hemoglobin are the dominant chromophores, while lipid and water are essentially invisible. With respect to microscopic optical imaging modalities, reflectance confocal microscopy (RCM) provides an alternative to biopsy. However, conventional RCM is limited to depths of 200-350 µm, and therefore deeper lesion margins may be missed and disease processes that extend beyond the epidermis are inaccessible. The short wave infrared (SWIR) spectral range (900-1700 nm) extends beyond the reach of Si-based detectors, and is characterized by increased water and lipid absorption, and decreased melanin absorption. In recent work we found that noninvasive SWIR imaging provides high contrast visualization of cutaneous inflammation that can be used to derive quantitative measures of inflammation that decrease linearly with disease progression. These considerations inform the central hypothesis that SWIR imaging will support objective assessment of inflammatory and neoplastic skin diseases regardless of skin pigmentation. In Aim 1 we will develop a macroscopic SWIR imaging modality and test its ability to assess disease severity in representative chronic inflammatory conditions in patients with diverse skin types. In Aim 2 we will develop a microscopic SWIR imaging modality and test its ability to surpass existing optical imaging depth limitations and also to differentiate benign from malignant lesions. These aims will be achieved through technical expertise in the Shmuylovich lab, with preclinical validation in skin mimicking phantoms followed by pilot studies in human subjects. This work represents a paradigm shift in cutaneous imaging whereby novel research-grade infrared imaging will be clinically translated to quantitatively assess disease across the full spectrum of diverse skin types, and at length scales ranging from whole body photography to virtual histology. These new technologies will have the potential to provide all patients with access to accurate diagnosis and management of skin disease and improve treatment outcomes.

NIH Research Projects · FY 2025 · 2021-09

Abstract The overall objective of this proposal is to answer two fundamental questions on DNA metabolism: 1) how DNA replication progresses timely and efficiently at hard-to-replicate sites; and 2) how the large nucleoprotein complexes that comprise telomeres, the end of linear chromosomes, are assembled and regulated. The first question emerges from our ongoing mechanistic studies on the conserved Pif1-family of helicases. Our work highlights the fundamental role that these helicases have in facilitating DNA replication at both secondary DNA structures and protein barriers. Going forward, we seek to address the following: what mechanisms couple the helicase activity to DNA synthesis by polymerases; how this synergy leads to removal of obstacles imparted by protein barriers encountered in the nucleus or in mitochondria; whether removal of obstacles is a general property of this class of helicases. The second question is novel and is based on our recent finding of new properties of both yeast and human telomere DNA binding proteins; namely, the ability of these proteins to interact with telomeric DNA repeats in alternative modes and to facilitate DNA condensation. Our goal is to test whether these properties play a role in establishing telomere length discrimination and accessibility. We tackle these problems by using biochemically reconstituted systems and employing a multipronged approach that integrates ensemble biochemical and biophysical techniques with single-molecule methods, thus providing unparalleled access to the behavior of individual molecules.

NIH Research Projects · FY 2025 · 2021-09

Type 2 diabetes mellitus (T2D) is a significant public health problem affecting ~30 million American. Obesity, insulin resistance, insulin deficiency (β cell dysfunction) and dysglycemia all precede the diagnosis of T2D and are known to promote inflammation and ultimately lead to microvascular complications. More recently, research has identified brain-related complications in adult-onset T2D, including reduced regional brain structure and function, impaired cognition, and increased lifetime risk for Alzheimer’s disease. Alarmingly, an increasing number of children and adolescents are being diagnosed with T2D, likely due to the growing prevalence and earlier onset of obesity. Youth-onset T2D appears to have a more aggressive course than adult-onset T2D, with earlier onset and more rapid progression of microvascular complications. In addition, studies of youth with obesity and youth-onset T2D have reported robust differences in regional brain structure and cognition, suggesting that brain effects may follow the same aggressive course as the more typical vascular complications. Unfortunately, little is known about the factors associated with poor brain structure and function in youth with T2D. To address this critical gap in knowledge, we propose to study youth across the spectrum of body mass index (BMI) and metabolic dysfunction. This approach will allow us to disentangle the relationship of key features of T2D risk (e.g. obesity) with intermediary physiologic changes that pose a risk for the brain (e.g. insulin resistance, inflammation, β-cell dysfunction and dysglycemia) that may lead to reduced brain structure and function in T2D. We will determine which of these factors are most associated with differences in brain structure and function among groups, over time, and how these effects differ from normal neurodevelopment. Given that the disease occurs at a time when brains are undergoing dramatic developmental processes, the aggressive nature of youth-onset T2D progression and complications in other organ systems, these results may provide guidance and justification for longer follow-up, interventional or mechanistic studies and have important clinical implications.

NIH Research Projects · FY 2025 · 2021-09

PROJECT ABSTRACT In response to PA-20-144 calling for innovations in HIV prevention, testing, adherence and retention to optimize HIV prevention and care continuum outcomes, we examine the impact and cost-effectiveness of an innovative multilevel intervention combining Multiple Family Groups (MFGs) for HIV stigma reduction (MFG-HIVSR) with a family economic empowerment (FEE) intervention versus a group-based HIV stigma reduction for Educators (GED-HIVSR) combined with MFG-HIVSR plus FEE (hereafter M-Suubi) on HIV treatment adherence and engagement in care among school-going adolescents living with HIV (ALHIV) in Uganda. HIV stigma remains a formidable barrier to HIV treatment adherence among adolescents in Uganda, contributing to low rates of medication adherence and viral suppression (less than 50%) and high attrition from HIV treatment services. ALHIV experience HIV stigma (internalized, anticipated and enacted) in various settings, including families and schools, the most important developmental contexts that should otherwise be supportive of their development and wellbeing. One of the unique features about education in Uganda and other countries in Sub-Saharan Africa is the high proportion (over 60%) of school-going adolescents enrolled in boarding secondary schools – which represent a form of parental opt-in institutionalized care. ALHIV in schools are more disadvantaged and have lower levels of HIV treatment adherence due to high levels of HIV stigma within schools, rigid school structures and routines, lack of adherence support and food insecurity. Within families, HIV stigma is perpetuated in various forms including discrimination and violence, often due to unfounded fears of infection—hence undermining the quality of family relations and supports for ALHIV. Building on our research and current evidence on HIV stigma reduction, we propose a multi-level three-arm cluster randomized study (M-Suubi) with the following specific aims: Aim 1: Examine the impact of M-Suubi on HIV viral suppression (primary outcome); and adherence to HIV treatment (keeping appointments, pharmacy refills, pill counts), and retention in care (secondary outcome); Aim 2: Examine the effect of M-Suubi on HIV stigma (internalized, anticipated and enacted), with secondary analyses to explore hypothesized mechanisms of change (e.g. depression) and intervention mediation; Aim 3: Assess the cost and cost-effectiveness of each intervention condition; and Aim 4: Qualitatively examine: a) participants’ experiences with HIV stigma, HIV treatment adherence, and the intervention; and 2) educators’ attitudes towards ALHIV, experiences with GED-HIVSR, and program/policy implementation post-training. The study will enroll 840 ALHIV recruited from 42 schools located within the greater Masaka region, heavily affected by HIV (prevalence 12% vs 7.3% national average). M-Suubi will be provided for 20 months, with assessments at baseline, 12, 24 and 36 months. Findings may inform combination intervention efforts to optimize HIV treatment outcomes and engagements in care among ALHIV.