University Of Michigan At Ann Arbor

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT Goal: The proposal outlined in this career development award (CDA) will provide Dr. Amarbir S. Gill with the protected time and training needed to develop into a successful, independent clinician-scientist. Dr. Gill's long- term career goal is to develop a translational research program that employs advanced molecular laboratory techniques to elucidate effects of environmental exposures on sinonasal epithelial barrier dysfunction in chronic rhinosinusitis (CRS) and identify key management strategies. Training: To achieve his goal, Dr. Gill has assembled a mentorship team that takes advantage of deep expertise in epithelial pathobiology, environmental exposures, label-free imaging, and bioinformatics at the University of Michigan, and has a strong track-record of mentoring K awardees to independence. Research: CRS is a debilitating inflammatory disease of the upper airway that affects up to 12% of the U.S. population. People with CRS battle daily nasal congestion, smell loss, nasal drainage, facial pain, as well as chronic fatigue, and sleep and cognitive dysfunction. The quality-of-life (QOL) impairment associated with CRS is on par with that of coronary artery disease, Parkinson's disease, and end stage renal disease. Despite its wide prevalence and significant morbidity, CRS remains an under- researched disease with limited effective treatment options. Available therapies fail to help more than 250,000 patients annually who then undergo sinus surgery, underscoring a critical need for improved knowledge of underlying molecular mechanisms driving disease pathophysiology. To this end, recent investigations have demonstrated that particulate matter (PM) exposure can increase the risk of developing CRS, while also impacting disease severity and surgical outcomes. We have shown these observations to be consistent for small, but not large PM particles. The present proposal seeks to understand the differential epithelial penetration and pathogenic potential of small vs. large PM particles, while identifying the role of sinonasal tight junctions (TJs) in propagating PM-mediated dysfunction of the sinonasal epithelial barrier in CRS. Our central hypothesis is that compared to large PM, smaller PM particles exhibit better sinonasal tissue penetration, allowing them to enter epithelial cells and initiate epithelial barrier dysfunction by downregulating key TJs, such as claudin proteins. To study this hypothesis, we will innovatively adapt fluorescence lifetime imaging microscopy (FLIM) – a label-free imaging tool that leverages differences in the autofluorescence spectra of PM and epithelial cells to track the movement of small and large PM particles – to in vitro air-liquid interface cell cultures and sinus biospecimens from individuals with CRS with nasal polyps, the most severe form of CRS. In doing so, we will define mechanisms facilitating size-dependent PM modulation of disease, uncover potential therapeutic targets, and inform novel management strategies.

NIH Research Projects · FY 2025 · 2025-09

ABSTRACT Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or meaningful treat- ments. ALS is mostly sporadic, and for most, evidence supports a gene-time-environment hypothesis of ALS triggered by a confluence of nonmodifiable risk factors (age, sex, and genetics) and modifiable risk factors from the exposome (a person’s cumulative lifetime exposures). Exposures imprint on the epigenome via DNA meth- ylation (DNAm) marks, which change with age, modifying cellular response to environmental insults. Compre- hensive knowledge of all these factors is needed to develop personalized prevention strategies for those at highest risk and personalized therapeutics for those already living with ALS. We reported that biofluid- and sur- vey-based exposure measures, summarized by environmental risk scores (ERS), strongly impact ALS risk and progression, adjusted for genetic background. We also recently discovered that epigenetic age acceleration, calculated from DNAm, is associated with ALS risk and exposures. However, the full spectrum of nonmodifia- ble and modifiable risk factors resulting changes in the epigenome are incompletely characterized. There is a critical need to fill this gap and determine how these factors impact ALS risk and the epigenome, leading to a better understanding of disease etiology, which can inform future precision strategies to prevent ALS. Our long-term goal is to leverage knowledge of the ALS exposome to inform personalized ALS prevention and therapeutic strategies. Our current objectives are to (i) comprehensively assess the exposome in our Michigan cohort and determine how it associates with ALS risk and survival; and (ii) gain insight into disease etiology by examining the intersection of the exposome with the ALS epigenome and transcriptome. Our central hypothe- sis is that ALS cases will have higher ERS scores, which correlate with epigenetic and transcriptomic changes and ALS risk and survival. In Aim 1, we will leverage targeted and untargeted exposomics in biofluids to identi- fy toxicant exposures that associate with ALS risk and survival and determine the effects of age, sex, and pol- ygenic risk on these outcomes. In Aim 2, we will identify geospatial- and survey-based exposomic measures linked to ALS risk and survival, and evaluate how they associate age, sex, polygenic risk, and biofluid toxicant measures. Finally, in Aim 3, we will characterize exposome-related DNAm and transcriptomic signatures and their association to ALS risk and survival. Completion of these studies will identify ALS-relevant exposome fac- tors that associate with to disease risk and survival via DNAm and consequent transcriptomic changes. These results will have important translational impact by lending insight into ALS etiology and identifying exposomic factors that most impact ALS risk and survival, thereby making the first critical steps towards personalized risk prediction and prevention strategies in ALS by exposure mitigation.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Specialized neurons in the nucleus of the solitary tract (NTS) receive and integrate gastrointestinal signals of ingestion along with other relevant interoceptive parameters (e.g., gut irritants and infection) to restrain food intake. NTS neurons that respond to gut irritants or systemic infection promote nausea associated behaviors and sickness responses, and consequently reduce food intake; NTS neurons that track ingestion restrain food intake to promote satiety. It is important to understand the specific types of NTS neurons that respond to these distinct stimuli, and disentangle NTS neurons that promote satiety from those that promote nausea or sickness responses. The broad goal of this proposal is to understand how systemic infection and feeding converge on NTS neurons to reduce food intake. The previous integration of mouse NTS snRNA-seq with human GWAS body mass index (BMI) data predict that a novel NTS neuron population marked by expression of Eya1 (NTSEya1 neurons) plays crucial roles in the control of food intake and body weight. In silico data from others predict NTSEya1 neurons may promote sickness responses to lipopolysaccharide (LPS) administration, a model of systemic infection. To understand the function and mechanisms of action for these cells, we generated Eya1Cre mice with which to manipulate NTSEya1 neurons and test each in silico prediction. Our preliminary data suggest activating NTSEya1 neurons reduces food intake and body weight without promoting conditioned taste avoidance, a nausea associated behavior. Silencing NTSEya1 neurons leads to hyperphagic obesity. In addition, these neurons are activated in response to refeeding and LPS, suggesting that NTSEya1 neurons may restrain food intake in response to two distinct stimuli. This proposal will test overall hypothesis that NTSEya1 neurons reduce food intake and promote satiety without causing sickness responses. To discover the physiological, and potential pathophysiological roles of NTSEya1 neurons, we will: (1) Determine if activating NTSEya1 neurons are sufficient promote sickness responses, (2) determine if NTSEya1 neurons are necessary for the anorectic response to LPS, (3) define the structure of NTSEya1 neuron projections and (4) identify the downstream projections of NTSEya1 neuron important to promote satiation. At the conclusion of these studies, we will have defined the roles NTSEya1 neurons play in the control of food intake, energy balance, and sickness responses. Thus, we will contribute to the broader understanding of how brainstem systems integrate multiple interoceptive parameters to restrain feeding, potentially revealing novel targets for the therapy of obesity.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Multiple adverse social risks (e.g. food and/or housing insecurity, transportation challenges, social isolation) and out-of-pocket, disease-related expenses are key reasons 1/3 of people with diabetes have high A1cs. Through our CareAvenue mHealth intervention (R01DK116715), we enhanced our original intervention of screening for social risks and connecting people with diabetes to resources by adding features for observational learning, autonomy support, action planning, and self-monitoring. We observed low engagement with the intervention and effectiveness for only 5% of participants. Social support interventions (e.g. peer support, financial navigators, social workers) are more effective in addressing unmet social needs and self- care challenges than stand-alone technology tools. However, these strategies are more resource- and labor- intensive. Rather than dispensing the same fixed package of treatment components to all patients, an adaptive approach can conserve resources by initiating autonomy-supportive treatment, and stepping up treatments for those with suboptimal uptake. This R01 builds on the productivity, infrastructure, and investment of our prior work to address key knowledge gaps for uptake of social care assistance to improve disease outcomes. The overarching goal of this application is to identify what type of supportive components are optimal for addressing unmet social needs and diabetes self-care, and which of five adaptive sequences of treatment results in better outcomes. We will recruit 594 people with diabetes who have high A1cs, unmet social needs, and want assistance with their needs. We will use a Sequential Multiple Assignment Randomized Trial (SMART) design with 12-month follow-up to conduct this research. In Aim 1, we will determine which of five adaptive intervention sequences is optimal for reducing A1c compared to social needs app only (usual care): 1) App + peer support; 2) App + social worker; 3) App + technology-supported financial navigation; 4) App + peer support + social worker; 5) App + peer support + technology-supported financial navigation. In Aim 2, determine which of two augmented treatment adaptations is optimal for reducing A1c among non-responders: social worker or technology-supported financial navigation. In exploratory Aim 2A, we will identify patient-level moderators of treatment effect to inform personalized, resource-efficient protocols. In Aim 3, we will estimate cost-effectiveness of five adaptive intervention sequences. We hypothesize that at 12 months, App + Peer Support augmented with technology-supported financial navigation will produce superior outcomes (reduced A1c, uptake of social care resources) relative to the 4 other adaptive interventions. The result of this SMART study will be an optimized, adaptive intervention to improve the health and social wellbeing of people with diabetes by determining the most effective intervention strategies.

NIH Research Projects · FY 2025 · 2025-09

Step-therapy is a utilization management strategy where insurers implement a tiered treatment pathway, and patients and physicians must obtain approval for restricted therapies by documenting failure of prior steps. Step therapy has been under increasing scrutiny because it may hinder access to necessary care. One prevalent syndrome where step therapy requirements are widely utilized, and impede access to patient-centered care, is overactive bladder (OAB). Alternatives to step therapy, including patient-centered treat-to-target approach, where therapy is determined based on severity of symptoms, have replaced step therapy for many chronic conditions. However, these patient-centered strategies have not been explored in OAB. OAB is characterized by urinary urgency, frequency, nocturia and/or urgency incontinence and affects up to 1/3 of Americans, primarily older women. While many therapies are available, currently patients and clinicians must navigate clinical tradeoffs and policy barriers associated with non-evidence-based step therapy that make treatment complex. Recent updates to national guidelines have admitted that there is no evidence to support the need for people with OAB to proceed with treatment using a stepwise approach. However, many payers require step-therapy for both medication coverage and procedural coverage of effective, minimally invasive therapies. The overarching aims of this proposal are to clarify the impact of step therapy as well as to evaluate alternative strategies in OAB care. First, we will use a nationally funded, multi-institutional electronic health record (EHR) dataset and a validated computational phenotype for overactive bladder to identify factors associated with update of minimally invasive therapies for OAB care (Aim 1). Next, we will quantify the impact of insurance provider on the utilization of these therapies using a regression discontinuity design (Aim 2). Findings from this study directly address the NIDDK mission to support clinical research to improve urological health. Specifically, it will allow patients, clinicians and policymakers to data to understand the impact of current step therapy policies, provide data for crafting exemption policies and support future trials evaluating practice policies and guidelines for minimally invasive OAB therapies.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Specific deficiency of fat tissue, or lipodystrophy, leads to progressive metabolic and endocrine disorders such as high triglyceride levels, insulin resistance, and ectopic steatosis especially in the liver. If the fat loss is generalized, patients also display leptin deficiency. Familial partial lipodystrophy (FPLD) is a primary cause of lipodystrophy syndromes in many affected individuals. In this form of lipodystrophy, fat loss is partial – affecting the limbs and the subcutaneous compartment of the trunk while adipose tissue in the face and neck as well as visceral fat depots are preserved and undergo compensatory hypertrophy. While these hypertrophic depots also display inflammation and scarring and would become insulin resistant over time, these depots preserve some ability to produce and secrete leptin. Consequently, patients with FPLD tend to have normal or even elevated endogenous leptin levels, which may be associated with decreased efficacy of exogenous leptin replacement therapy. Thus, although metreleptin is approved as therapy for generalized lipodystrophy, FPLD patients represent a high unmet medical need because there is no FDA-approved therapy for FPLD in the US. Nevertheless, because patients with FPLD have markedly increased appetite, this disease is characterized by a mismatch between their large caloric intake in the face of limited adipocyte storage capacity. We propose to test the hypothesis that patients with FPLD would derive clinical benefit if treated with a drug that decreased food intake. Tirzepatide is a dual incretin (activating receptors for both GLP1 and GIP) that induces an unprecedented magnitude of weight loss in patients with obesity and overweight status. In addition, tirzepatide is reported to enhance glucose-stimulated insulin secretion, enhance storage of triglyceride in white adipose tissue, and increase insulin sensitivity in skeletal muscle. These pleiotropic pharmacologic activities offer potential benefit for patients with FPLD. This application proposes a prospective randomized clinical trial testing the efficacy of tirzepatide in improving body weight and fat mass, adipocyte inflammation, metabolic perturbations such as hyperglycemia and dyslipdemia, hepatic steaotosis and hepatic stiffness (fibrosis). We will also collect information on important safety parameters and some parameters of patient reported outcomes. This rigorously designed trial offers potential to address the unmet medical need of patients with familial partial lipodystrophy and will shed light on pathophysiology of familial partial lipodystrophy as well as novel insights on the crosstalk among gut hormones (GLP1 and GIP), adipose tissue, and appetite regulation by the brain.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Thoracic aortic aneurysm and dissection (TAAD) are among the major causes of morbidity and mortality in the United States. Thoracic aortic aneurysms (TAAs) show sex differences in growth rates and outcomes for unknown reasons. Despite the well-established role of genetic predisposition in TAA formation, the majority of TAA patients do not have a family history of the disease or syndromic features, with disease presentations at older ages. Causes of sporadic TAA are insufficiently understood. Current TAA treatment is limited to surgical intervention due to inadequate understanding of the disease pathogenesis specific to humans. Ambiguities still exist regarding the spatial remodeling in human TAA tissue, leaving the disease-causing and protective changes unexplored. Therefore, molecular and functional characterization of the cells constituting human TAA tissue is vital in understanding the mechanistic underpinnings of sex differences in TAA formation and outcomes. Using high-resolution spatial profiling, we discovered spatially-distinct CARTPT (CART prepropeptide) expressing cells predominantly in male sporadic ascending TAAs. Our hypothesis is that sexually-dimorphic CART signaling mediates aortic wall remodeling and medial calcification in male TAAs. Medial calcification reduces vessel compliance and increases arterial stiffness, but whether it is a friend or a foe (or both) in TAA pathogenesis remains to be determined. The aims of proposed project are the following: (1) Define the molecular triggers and consequences of CART signaling in aortic smooth muscle cells in vitro, and (2) Determine the role of sexually dimorphic signaling mechanisms in TAA pathogenesis in vivo. To investigate the proposed aims, we will utilize a toolset tailored for basic human research including human derived smooth muscle cells, bioengineered human vascular grafts and high-content single molecule spatial transcriptomics profiling of surgically resected TAA samples as reference points. Achievement of the proposed studies will resolve the regulation and the functional relevance of sexually dimorphic cells and signaling pathways in human TAAs with an emphasis on CART signaling, determine how these sex differences influence TAA outcomes and provide high resolution spatial maps of male and female TAAs.

NIH Research Projects · FY 2025 · 2025-09

Project Abstract Human pluripotent stem cell (hPSC) derived human intestinal organoids (HIOs) are a powerful model since they possess many lineages, including epithelium, smooth muscle, neurons, and endothelial cells; however, HIOs lack immune populations, which are key to studying the development and maturation of the intestine. Since early HIOs possess mesoderm, I tested the hypothesis that this population may respond to cues, such as BMP signaling, that induce immune cell differentiation. Preliminary data supports a role for BMP signaling as an inductive cue for CD45+ immune cells within small intestinal HIOs. This proposal will test the hypothesis that early HIOs retain plasticity soon after intestinal patterning and have mesenchyme-specific responses to BMP signaling, which leads to differentiation of the mesenchyme into hemogenic endothelium that will further differentiate into functional immune lineages. I aim to interrogate the mechanism by which BMP4 induces immune-like cells, through a hemogenic endothelium intermediate, in HIOs, including the cell intermediates required for immune cell differentiation. Secondly, I aim to investigate the lineages of immune cells present and functionally evaluate the immune-like cells in the HIOs.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Intestinal iron absorption is the major mechanism of iron delivery in mammals. Cellular or systemic iron deficiency can lead to dysregulation of metabolic pathways and anemic disorders. However, excess iron such as in hemochromatosis can lead to tissue injury and cell death. Therefore, systemic and cellular iron levels are tightly regulated. Due to the reactive nature of iron, iron is stored in ferritin, a protein capable of binding up to 4500 iron atoms. In conditions of high iron demand or deficiency, ferritin is degraded, releasing iron for cellular and systemic utilization. Ferritin is a critical protein that controls cellular iron, intestinal iron absorption, and oxidative metabolic pathways. Moreover, ferritin is essential in nutritional immunity, sequestering iron from microbial pathogens that require it for growth. Notably, in germ-free (GF) or antibiotic treated (Abx) mice, we observed a significant decrease of ferritin expression both locally in the intestine and in peripheral tissues. These findings indicate a dynamic regulatory role of host cells in modulating their iron storage pathways in response to commensal bacteria. We demonstrated that ferritin levels were restored in germ-free (GF) or antibiotic treated (Abx) mice upon treatment with microbial metabolites. This data suggests a metabolic exchange between commensals and epithelial cells that drives the ferritin response. Specifically, a synthetic community composed of 11 gram-negative and gram-positive commensals successfully restored intestinal ferritin expression in germ- free mice. Despite assessing several canonical mechanisms of ferritin regulation, including mRNA levels, autophagic degradation of ferritin (ferritinophagy), and hypoxic pathways, no changes were observed. We found altered activity of iron regulatory proteins (IRPs) in germ-free mice compared to wild type. IRPs are RNA binding proteins that regulate ferritin translation. I hypothesize microbial metabolites suppress mRNA binding activity of IRPs leading to an increase in host ferritin expression. However, studying microbial and intestinal epithelial cell interactions in vitro presents challenges due to the anoxic conditions required to grow anaerobes. Our ongoing work aims to elucidate the precise mechanism of microbial regulation of host ferritin levels and identify microbes and metabolites capable of inducing ferritin expression. I will test this hypothesis through two aims. Aim 1 will identify microbe(s) and their metabolites capable of inducing ferritin expression. This will be done using an asymmetric co-culture system that allows us to culture anaerobic bacteria adjacent to primary intestinal epithelial cells in an environment mimicking the intestine. Aim 2 will determine the cellular mechanism of microbial regulation of host ferritin. I will utilize conditional knockout mice for IRPs to determine if ferritin translation is regulated by microbes. Understanding the mechanism of microbial integration into host ferritin regulation is crucial for defining precise mechanisms by which microbiota regulate intestinal iron absorption, iron related disorders and in microbial pathogenesis.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Viruses are obligate intracellular parasites that cannot replicate or survive outside of a host. To produce new viral progeny, they must first gain entry into their target host cells where they hijack various cell biological processes to complete their life cycles. The overall goal of my research program is to determine the molecular mechanisms required for successful viral entry and to identify the host-pathogen interactions governing this essential step in infection. In general, viral entry requires receptor binding at the host cell surface, internalization, and subsequent transport of the virus through the endomembrane system towards its site of replication. Most viruses with DNA genomes replicate in the nuclei of infected cells and, consequently, must cross the nuclear envelope to access host DNA replication machinery. How viruses penetrate this physical barrier to enter the nucleus is a major gap in understanding that will be addressed by this research proposal. We are particularly interested in understanding how viruses with oncogenic (cancer-causing) and oncolytic (cancer-treating) potential enter cells to establish infection. Over the next five years, we will investigate the general entry mechanisms of two DNA viruses—the oncogenic Merkel cell polyomavirus (MCPyV) and the oncolytic LuIII parvovirus. Using a combination of molecular biology, biochemistry, and high-resolution microscopy techniques, this R35 proposal will determine (1) how MCPyV and LuIII target to and cross the nuclear membrane, and (2) the host factors that are required for this process. Interestingly, both viruses appear to use non-canonical nucleocytoplasmic transport mechanisms to reach the nucleus. Therefore, these research findings will increase our fundamental understanding of viral nuclear entry mechanisms to advance the diagnosis, treatment, and prevention of virus-associated disease. Moreover, the transport of molecules into and out of the nucleus is often dysregulated in unrelated human disorders, so this work also will provide additional insight into cellular nucleocytoplasmic transport mechanisms that are more broadly relevant to human health. Overall, these studies will lay the foundation for a productive and well-established research program investigating the cellular entry mechanisms of these and other DNA viruses while increasing our understanding of the basic biological pathways with which they interact.

NIH Research Projects · FY 2025 · 2025-09

Diabetes and dementia have an unexpectedly high rate of comorbidity: individuals diagnosed with diabetes have a 1.4–2.2-fold higher risk of dementia than those without diabetes. The healthcare burden of these diseases also differs by race/ethnicity backgrounds (higher in non-Hispanic Black individuals) and by biological sex (higher in men for diabetes but women for dementia). Epidemiological studies have proposed shared risk factors, metabolic disorders, comorbidities, and genetics to explain this relationship but human research in assessing molecular mechanisms is challenging due to environmental influences. Nevertheless, Dr. Litkowski, the primary investigator of this proposal, provided the first causal genetic evidence between diabetes and dementia in her work with the Million Veteran Program. The long-term goal of this project is to elucidate the mechanism by which impaired glucose metabolism leads to cognitive decline. This proposal integrates human findings with causal testing, and preclinical interventions using a unique discovery pipeline in the Alzheimer’s disease mouse panel (AD-BXD), a genetically diverse model incorporating AD loci known to predict cognitive decline in human populations. The objective is to 1) confirm the genomic region Prdm10 leading to declines in postprandial glucose metabolism in AD susceptible females fed a high fat high sucrose diet and 2) evaluate an anti-diabetes drug, miglitol, on strains deemed to be susceptible to cognitive decline. The hypothesis is that genetic (Prdm10) and pharmacological (miglitol) interventions will modify cognitive resilience. This project will be conducted at the University of Michigan (UM), the #2 public research university (and #4 in overall rank) in the U.S., according to the National Science Foundation, with annual research expenditures of ~$1.77 billion dollars and ~$970 million dollars in federally-sponsored research in 2022. UM is a premier biomedical research institution with first-rate core facility services for its researchers, specifically the Bioinformatics Core, Metabolomics Core, and Transgenic Animal Core, relevant to this proposal. The primary investigator (and trainee) of this proposal (Dr. Litkowski) will have access to the extensive facilities and equipment of the Dr. Kaczorowski Laboratory, including a state of-the-art behavioral suite outfitted for cognitive testing (passive avoidance, Y-maze, contextual fear), a surgical suite, and 2 large animal housing rooms. The proposal includes a detailed training plan for Dr. Litkowski mentored by her sponsor, Dr. Kaczorowski, and co- sponsor, Dr. Bridges, both of whom have previously mentored postdoctoral fellows to become independent investigators. The training plan establishes a timeline for achieving data acquisition proficiency to assess glucose/insulin metabolism and cognition, and for developing and implementing innovative reverse genetics approaches. In summary, successful completion of these aims will define Prdm10 as a causal gene, and miglitol as an effective intervention leading to improved glucose metabolism and resilience to AD-related cognitive decline while at the same time preparing Dr. Litkowski for a career as an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

Abstract Type 1 Diabetes (T1D) represents a significant global health challenge, characterized by complex interactions between genetic, environmental, and phenotypic factors. The NIDDK has funded numerous T1D initiatives over the years, generating vast scientific data across multiple modalities. However, there is still no system to fully leverage these large, multimodal datasets to build an AI model to facilitate data-driven knowledge discovery. Advances in AI and multimodal data analysis now offer opportunities to revolutionize our understanding of complex diseases like T1D. In this proposal, we will build MAI-DK (Multimodal AI - from Data to Knowledge), a system that converts multimodal data into a knowledge-generating machine. This system will provide a systematic overview of the molecular mechanisms controlling T1D progression across multiple organs. Achieving this goal will require an interdisciplinary team with expertise in genetics, genomics, pancreas and islet biology, biostatistics, machine learning, AI systems, and ethics. We have assembled a ten-MPI team with established, productive collaborations in these areas. The MAI-DK system will serve the diabetes research community, focusing on T1D pathogenesis and progression, and cater to clinical scientists, basic scientists, data scientists, and developers. To accomplish this, we propose five components: data modalities and QC, multimodal AI models and system, ethics, co-design, and stakeholder engagement. Completing these components will make MAI-DK an attractive destination for T1D research communities, accelerating progress in understanding T1D and informing transformative changes in diabetes prevention and care.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Chronic pelvic pain (CPP) affects 15-20% of U.S. women of reproductive age and is linked to gynecological conditions, such as endometriosis and dysmenorrhea. Accumulating evidence suggests that altered pain processing in the brain (i.e., nociplastic pain, which predicts persistent comorbid pain disorders and treatment resistance) is an underlying cause in some cases. Despite the emergence of CPP during puberty, very little is known about how nociplastic pain manifests in adolescents, and neuroimaging studies are scarce. Furthermore, drastic changes to the CNS occur during puberty, with substantial heterogeneity that usually goes unexamined. Heterogeneity also characterizes the etiology, presentation, and neuroendocrinology of CPP in dysmenorrhea and endometriosis in adults. Thus, empirical research that centers on development and variability is necessary for meeting the unique needs of individuals who walk into clinics. The overarching goal of this proposal is to map the personalized neural variability underlying reproductive health-related CPP, informing important clinical outcomes by identifying risk factors and eventually improving early detection and intervention. The specific aims are to: 1) examine personalized neural networks involved in gynecological CPP in adult females; 2) identify individualized trajectories of dysmenorrhea pathogenesis, before and after menarche, and consequently, predict new pain onset in adolescence; and 3) conduct a longitudinal neuroimaging study to determine the heterogeneous neurobiological correlates of developing one or more Chronic Overlapping Pain Conditions from dysmenorrhea alone in adolescence. The main goals of the K99 portion are focused on providing the candidate innovative training in: a) pain phenotyping and neurobiology; b) idiographic methods, including developmental trajectories and personalized neural network analyses; and c) pubertal assessment. The K99 phase will take place at the University of Michigan (UM), under the mentorship of Dr. Adriene Beltz, a world-renowned expert in behavioral neuroendocrinology and developmental idiographic science. Drs. Chelsea Kaplan, Andrew Schrepf, and Daniel Clauw, internationally recognized experts in chronic pain mechanisms and neuroimaging, and Dr. Sawsan As-Sanie, an expert in clinical management of endometriosis and other CPP conditions, will also be mentors. The UM is an ideal training environment because of its extensive interdisciplinary resources, including the Department of Psychology, Chronic Pain and Fatigue Research Center, and the Department of Obstetrics and Gynecology. UM faculty mentors are leaders in their fields, have a history of successful collaboration, and are dedicated to data sharing and training. Upon completing the K99 portion, the candidate will be well-prepared to transition into an independent tenure-track faculty position studying neurobiological correlates and individualized mechanisms underlying the development of CPP conditions during adolescence. This work is crucial for women’s well-being, and highly novel in its focus on heterogeneity in the adolescent development of pain-related reproductive health disorders.

NIH Research Projects · FY 2025 · 2025-09

Despite advances in CRC care over the last two decades, differences in the incidence and mortality from colorectal cancer (CRC) between younger and older patients in the U.S. continue to persist.  Existing research highlights differences in the incidence, stage at diagnosis, survival rates, and access to treatments for younger patients with CRC. Younger survivors may also have distinct survivorship concerns compared to their older counterparts. However, the specific needs and priorities of these young survivors remain underexplored. In this study we will utilize Michigan Cancer Surveillance Program (MCSP) Clinical Registry and Michigan Value Collaborative (MVC) Administrative Claims Data to examine contemporary survivorship patterns in younger colorectal cancer survivors. We will specifically examine rates of National Comprehensive Cancer Network (NCCN) guideline concordant surveillance among young colorectal cancer survivors, examining clinical and demographic factors associated with non-receipt of guideline concordant surveillance (Aim 1). We will then interview a cohort of young colorectal cancer survivors diagnosed with stage I-III CRC and underwent surgery from 2018-2022 (Aim 2). This study will yield much-needed population-based estimates of surveillance intensity and survivorship care needs young cancer survivors.

NIH Research Projects · FY 2025 · 2025-09

Abstract: Meaningful impact on the prevalence and incidence of substance use disorders (SUD) and HIV requires an array of evidence-based interventions that address individuals’ changing needs, circumstances, and strengths. Digital adaptive interventions (DAIs) that leverage advances in digital technologies such as mobile devices, wearable sensors, and artificial intelligence have shown great promise in improving outcomes for managing SUDs and preventing HIV, as well as for improving outcomes in other health domains. However, such interventions remain difficult and resource- intensive to build, and existing approaches to building them fail to support dissemination of best practices in both methodology and building the evidence base. In this project we will build and make available JustIn: a sustainable open source software (OSS) platform and community for advancing research in adaptive interventions. Within the scope of this project we will develop and release an initial version of JustIn that supports the majority of the functionality required to create existing state of the art adaptive interventions. Along with the initial release we will provide comprehensive documentation, tutorials, and working sample application code to bootstrap the adoption of JustIn for future intervention development. Following best practices for creating sustainable OSS communities, we will develop guidelines and procedures for integrating code contributions from the wider research community and implement governance structures that facilitate community input on features and priorities for future versions of JustIn. JustIn will democratize research to optimize digital adaptive interventions in SUD/HIV and other domains of health, allowing investigators to keep pace with emerging technologies and leverage them to produce radically effective and resource- efficient interventions. The growth in systematic development of novel digital adaptive interventions has the potential to reduce the burden of SUD/HIV and other chronic disorders.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Microsurgery is at the forefront of modern surgical practice. The quest for ultra-precise resection of invading tumors, durable anastomosis of severed neurovascular structures, and overall minimization of invasiveness has driven the scale of surgical maneuvers below a millimeter and firmly into the microsurgical domain. Applications of microsurgery abound, commensurate with the potential patient benefits, including tissue sparing, better margins, function restoration, and reduced pain. Uniting its diverse procedures is the defining characteristic of microsurgery and the source of its challenges: the sub-millimeter scale of surgical maneuvers and target tissues. The human motor and perception systems are ill-equipped to operate in this regime, and microsurgeons thus reach intrinsic limits of human performance and encounter shortcomings of surgical aids at microsurgical scales. Fortunately, advances in robotics, virtual reality (VR), and artificial intelligence (AI) promise to solve these fundamental challenges from manipulation, visualization, and workflow standpoints, respectively. These incredible advances and, in some cases, commercial products fail microsurgeons from the perception standpoint, however. The project therefore proposes to develop an adaptive perception system based on optical coherence tomography (OCT) to satisfy this unmet need in microsurgery. Specifically, adaptive perception leverages an AI-derived understanding of the surgical scene and its dynamics to build a high-fidelity 3D reconstruction from intelligently distributed image acquisition effort. Critically, OCT is the only live volumetric medical imaging modality routinely acquired during surgery and thus presents a unique opportunity in microsurgery for 3D surgical feedback. Leveraging adaptive perception to guide surgery for natural and artificial agents, this project will create an OCT-integrated robotic microsurgery system capable of autonomous and VR tele-microsurgery modes. Its core capabilities include an OCT-based adaptive perception system that predicts and responds to the microsurgeon’s or AI’s visualization needs, immersion of the microsurgeon in a life-size VR environment constructed from live volumetric OCT upsized 100-1000x to human scale, and AI-guided automated microsurgical suturing without fiducial markers. This project further proposes to compare this system to conventional techniques in simulated and ex vivo microsurgical procedures. This platform promises to empower microsurgeons to complete their tasks faster, commit fewer technical errors, require less training to reach proficiency, and enjoy improved ergonomics. These benefits would translate into the downstream patient benefits of improved outcomes, reduced anesthesia time, faster recoveries, and easier access to care for hundreds of thousands of microsurgery patients each year. These innovations and applications promise to transform microsurgery in ways that could not be envisioned even a few years ago.

NIH Research Projects · FY 2025 · 2025-09

Project Summary: Housing insecurity is a pressing public health problem: U.S. rates are the fastest-growing among older adults, one-third of whom spend more than 30% or 50% of their income on housing. Housing insecurity―defined as limited access to and availability of affordable, stable, safe, and adequate housing and neighborhoods― is a risk factor for numerous adverse health outcomes including chronic conditions, disability, and mortality. Although federal housing assistance is associated with improved health outcomes, current programs support only about one-third of income-eligible, cost-burdened adults aged 50+ who need assistance. Moreover, the relationship between housing assistance, housing insecurity, and adults' health and disability trajectories remains poorly understood. One unexplored area is how housing assistance and multiple dimensions of housing insecurity experienced during emerging adulthood, a critical life course period, relate to midlife and older adult health and disability trajectories. While previous research has examined effects of housing assistance and housing insecurity on health, studies often use cross-sectional or convenience samples, focus on a single rather than combined effects of multiple housing insecurity dimensions, and rarely include income-eligible older adults who do and do not receive housing assistance. Examining how previous housing histories relate to health and disability later in life will enhance our understanding of when housing assistance and housing insecurity matter most, and inform future housing interventions. The proposed K01 study leverages more than 50 years of nationally representative, prospectively-collected longitudinal life course data from the Panel Study of Income Dynamics to examine how midlife and older adult health and disability trajectories relate to housing assistance and housing insecurity experienced during emerging adulthood, a critical life course period. This project seeks to: (1) develop typologies of housing assistance and housing insecurity life course histories for midlife and older adults; (2) examine how trajectories of health and disability during midlife and older adulthood relate to previous histories of housing assistance and housing insecurity; and (3) assess the extent to which relationships between midlife and older adult health and disability trajectories and housing history typologies vary by neighborhood context. Complementing this research, a detailed training plan will build on the applicant's prior training in environmental psychology and architecture to include (1) aging, gerontology, and life course theory; (2) age-related disability, mobility, and physical function; (3) longitudinal survey data analysis skills, including latent variable modeling; and (4) external grant-writing skills. This integrated training will prepare the applicant for a successful independent research career focused on aging, housing, and neighborhoods. Findings from this proposal will generate critical insights concerning for whom, how, and when housing assistance and housing insecurity matter most for midlife and older adults' health and disability, inform future housing interventions, and elevate older adults' need for housing assistance.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The Panel Study of Income Dynamics (PSID) is a longitudinal, nationally representative survey of US families. The study began in 1968 and has collected 43 waves of data over 56 years on the original families and their descendants, and has added two major immigrant refresher samples in 1997–1999 and 2017–2019. PSID serves as a cornerstone for empirical social and behavioral research through its long-term measurement, over the life course and across generations, of economic, social, demographic, and health processes. The data are available free of charge to the research community, with outreach and support to new and established data users. These features have led PSID to become one of the most widely used social science data sets in the world. The study’s innovative design, broad content, and long duration have been central to understanding many key research and policy issues. The data support increasingly rich models of child and adult outcomes over the life course and across multiple generations of the same family. NICHD has co-funded the biennial Core PSID for each of the last 11 waves, from 2003 to 2023. This project builds upon this investment through the following specific aims: First, to collect data on three modules—family dynamics, fertility and newborns, and education—as part of the 2025 wave of the Core PSID survey. Second, to incorporate new measures of personal perceptions of unfair treatment due to demographic, social, and/or economic characteristics. Third, to process, document, and disseminate these data without charge to the research community while providing outreach and support to new and established data users. This project will make several major contributions. It will extend the longest-running household panel survey in the world, supporting new and up-to-date research on family dynamics, investments in children, and well-being over the life course, across generations, and over time. PSID data from 2025 will support research on the medium-term effects of the global pandemic of 2019 on behavior and well-being, and will facilitate future studies on how life course trajectories were altered by exposure to the pandemic. The rich data to be collected on education will support detailed analyses of the determinants of schooling decisions within the family context as well as the effects of these decisions on life course outcomes. Information from the newborn module will allow research on birth outcomes and on the consequence of birth outcomes and very-early life experiences on life course trajectories. Continuing data collection on the dynamic processes of family formation and dissolution, fertility, and living arrangements will allow researchers to understand the evolving complexity and circumstances of families in the U.S. The new data on personal perceptions of unfair treatment will immediately support innovative analyses of the social, economic, and contextual factors that shape these perceptions and will subsequently support research on their health and well-being consequences. Lastly, data user outreach and assistance will stimulate and support research using PSID and engage a new generation of scientists.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Management of anticoagulant medication, including for the management of low-risk pulmonary embolism, is challenging and frequently does not follow evidence-based guideline recommendations. On the one hand, up to 40% of all patients with an acute pulmonary embolism are at sufficiently low risk for complication that guidelines recommend outpatient management rather than hospitalization. However, this evidence-based practice is rarely utilized in the United States. On the other hand, when oral anticoagulants are prescribed for pulmonary embolism and other common thrombotic conditions, up to 25% of prescriptions do not follow evidence-based guidance and patients are exposed to unnecessary life-threatening risks of thromboembolism or bleeding. The research activities supported by this K24 award will complement Dr. Barnes’ ongoing NIH- funded R01 project that aims to improve the use of outpatient management of low-risk pulmonary embolism across 12 health systems in Michigan. This award also leverages Dr. Barnes’ current AHRQ R18-funded project that uses both electronic health record alerts and clinical pharmacists to improve safe- and evidence- based anticoagulant prescribing. While Dr. Barnes’ career goal is to develop, implement, and evaluate anticoagulation stewardship efforts, such as the ones being tests in his current R01 and R18 projects, he also is passionate about mentoring the next generation of patient-oriented researchers who focus on implementation science methodology. Dr. Barnes will leverage ongoing NIH R01 funded projects to expose his mentees to practical aspects of implementation science, including implementation mapping and qualitative assessments of implementation adaptation. Dr. Barnes will also focus on his own career development by gaining critical training in optimization trial design and advanced mentoring skills. Dr. Barnes has assembled a senior, diverse, and highly committed set of advisors to oversee his personal career development. He has also identified several channels for increasing his mentorship opportunities with clinician-scientists eager to develop as independent patient-oriented researchers who focus on implementation of evidence-based clinical medicine.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Steatotic liver disease (SLD) prevalence in just the United States is estimated to upwards of a quarter of the population. It ranges in presentation from simple Hepatic Steatosis without evidence of liver injury to development of inflammation and fibrosis (Metabolic Associated Steatohepatitis; MASH). Those individuals that develop MASH have increased risk of progression to advanced fibrosis and ultimately cirrhosis. Understanding the cellular and molecular mechanisms of the transition from simple steatosis to inflammation and fibrosis is needed. Recent work has shown that MASH leads to high level of hepatic and systemic ammonia. Moreover, there is mounting evidence that ammonia can contribute to the progression of MASH. Ammonia levels are detoxified through the urea cycle. The liver is the major site of ammonia detoxification and consistent with the increase in ammonia in MASLD, urea cycle genes are significantly decreased in MASLD. However, the molecular mechanism driving the metabolic reprogramming to alter liver ammonia is unclear. Preliminary studies have implicated the hypoxia-inducible factor (HIF) pathway in the development of liver steatosis and fibrosis. I show that activation of hepatocyte HIF2a (but not HIF1a) signaling decrease urea cycle genes, increases ammonia levels, steatosis, inflammation, and fibrosis. Therefore, I hypothesize that HIF2α exacerbates liver injury and dysfunction by disrupting urea cycle metabolism, resulting in steatosis, and promoting an inflammatory and profibrotic response. To test this hypothesis, I propose two Aims: 1) To understand how dysregulation of HIF signaling drives altered hepatocyte gene expression and metabolism. Hepatic nuclear factor (HNF)4α is the master transcription factor for the regulation of urea cycle genes. I will understand the crosstalk of HIF2α and HNF4α signaling. 2) Define if inhibition of HIF2α is a viable target for clinical intervention in the progression to advanced fibrosis/cirrhosis. Recently a clinically relevant on-target HIF2a, beltifuzan was approved for clear cell renal carcinomas. I will test the role of beltifuzan in preclinical models of MASH. The proposed research seeks to understand the role of HIF signaling in the development of steatosis and fibrosis that if left unabated can progress end stage liver disease and to the sequalae that drive the significant morbidity and mortality of the disease.

NIH Research Projects · FY 2025 · 2025-09

The University of Michigan and the Tissue Engineering and Regenerative Medicine International Society, Americas Chapter (TERMIS-AM) will be hosting and organizing the upcoming 2025 meeting of the TERMIS, Americas Chapter (TERMIS-AM) Conference and Exposition. Michigan is requesting support for travel awards for the Student and Young Investigator Section (SYIS) trainees to attend the full conference. The theme of the conference and the highlight of the meeting is “Next Generation Tools for Regenerative Medicine.” The preconference symposia will focus on strategies for accomplishing translational research and bringing regenerative medicine therapies to the clinic.

NIH Research Projects · FY 2025 · 2025-09

Why do we age and why do some of us stay healthy longer than others? Thanks to the development of shorter-lived aging model organisms, much has been learnt about the processes that contribute to our inevitable demise, and the tight connection between these processes and the development of age-associated diseases, including Alzheimer’s and Parkinson’s Disease. Our recent genetic and biochemical studies in C. elegans have revealed a novel longevity paradigm, triggered by early-in-life oxidative stress and mediated by the redox sensitivity of the histone 3 lysine 4 trimethylating (H3K4me3) COMPASS complex. We discovered that modulation of the H3K4me3 landscape in C. elegans led to the activation of at least two highly conserved longevity factors, heat shock factor-1 (HSF-1) and the starvation response regulator HLH-30 (TFEB in mammals), both of which essential for the lifespan-extending effects. Mechanistic follow-up studies revealed that the observed increase in lifespan depends on a non-canonical role of HSF-1 in modulating lipid homeostasis and fatty acid oxidation and involves a hitherto unknown regulatory interaction between HSF-1 and HLH-30. We found this interplay between transient ROS accumulation, changes in H3K4me3 levels and transcription factor activation to be conserved from C. elegans to mammalian cells and, most recently, to select tissues of post-weaned mice, which were exposed to oxidants for the first three weeks of their life. Based on these exciting data, and recent reports that in a range of different model organisms, including mice, early-in-life time windows exist in which lifespan can be set, we now propose that mild oxidative stress, when experienced at the right time in life, triggers a hormetic response that manifests itself through stable changes in the epigenetic landscape, gene expression and physiology. In this proposal, we will take a multipronged approach to determine the molecular mechanism by which ROS-mediated changes in developmental H3K4me3 levels promote lifespan extension in C. elegans (Aim 1), explore the long-term epigenetic and transcriptional changes that are elicited by short-term oxidative stress treatment in C. elegans as well as mitotic and postmitotic mammalian cells (Aim 2), and evaluate the molecular and physiological consequences of early-in-life oxidative stress treatment in mice (Aim 3). These studies have the clear potential to provide us with previously unknown mechanistic insights into how ROS-sensitive epigenetic circuits transform transient events in early life into long-lasting, and potentially universal, health- and lifespan extending processes. Our studies in C. elegans and cell culture models will furthermore serve us to guide parallel explorations in mice and provide new leads useful in the search for drugs that slow aging in mammals.

NIH Research Projects · FY 2025 · 2025-09

Abstract: It has been well documented that disease activity in inflammatory mucosal diseases including inflammatory bowel disease is linked to the transepithelial migration of neutrophils through the mucosal epithelium and ultimately into the intestinal lumen. Co-incident with massive luminal influx of neutrophils is uncontrolled release of toxic tissue damaging metabolites that contribute to mucosal and/or transmural injury including edema, loss of goblet cells, decreased mucus production, crypt cell hyperplasia, ulceration and crypt abscess formation. The complex signaling mechanisms that regulate neutrophil transepithelial migration and inflammatory function in mucosal tissues have not been fully elucidated even though excessive neutrophil activation is heavily implicated in disease pathology of IBD and other inflammatory disorders. Preliminary data identifies the tyrosine phosphatase CD45 as an important regulator of neutrophil intestinal infiltration and inflammatory function. This proposal will determine effects of neutrophil specific loss of CD45 on intestinal inflammation and repair using novel neutrophil specific CD45 deficient mice. We will also determine how CD45 phosphatase activity regulates neutrophil intracellular signaling pathways and inflammatory effector functions including migration, degranulation, and superoxide release. Finally using specific antibodies and glycan binding proteins we will determine how specific CD45 isoforms and glycoforms can be targeted to regulate neutrophil activation and inflammatory responses. Elucidating beneficial effects of targeting neutrophil CD45 phosphatase activity during colitis will help to develop novel strategies to ameliorate pathological intestinal inflammation.

NIH Research Projects · FY 2025 · 2025-09

Abstract Cellular and organismal homeostasis requires the integration of diverse environmental cues by cell signaling networks, the disruption of which contributes to pathological conditions. The kinase mTOR, which comprises the catalytic core of two distinct complexes (mTORC1 and mTORC2), responds to nutrients (amino acids (AAs); glucose), growth factors (EGF), hormones (insulin), and energetic stress to control fundamental cellular processes that impact cell metabolism, growth, proliferation, and survival. Not surprisingly, aberrant mTOR signaling contributes to diverse pathologic states including type II diabetes, cancer, and immune and neurodegenerative disorders. Despite the physiological importance of mTOR, major gaps exist in our understanding of mTOR regulation and function, in particular how mTOR communicates with other regulatory systems. Our research focuses on how non-canonical functions of the energy sensing kinase AMPK and the innate immune kinase TBK1 control mTOR signaling and function in cells and in mice in vivo. While it is well- established that AMPK inhibits mTORC1 during energetic stress, our prior work revealed that AMPK paradoxically i) phosphorylates mTOR on a site that promotes mTORC1 signaling and ii) supports mTORC1 signaling during prolonged amino acid (AA) deprivation, a stress condition that induces autophagy and liberates AAs, a nutrient that supports mTORC1 signaling in response to growth factors and hormones. These results reveal AMPK as a previously unknown sensor of cellular AA levels. Thus, this proposal will define how AMPK supports mTORC1 signaling and function in specific cellular contexts rather than universally opposing mTORC1, which would invoke a paradigm shift to the AMPK and mTOR fields. While TBK1 is best known to initiate host defense responses against microbial pathogens, TBK1 also contributes to tumorigenesis and glycemic control during obesity. How TBK1 mediates these non-canonical processes remains poorly defined, however. Our prior work revealed that i) TBK1 phosphorylates mTOR to promote mTORC1 and mTORC2 signaling in response to microbial-derived signals and insulin and ii) the TBK1-mTOR axis protects against hyperglycemia and insulin resistance in obese but not lean mice. Our preliminary results also indicate that AAs increase TBK1 activity, revealing TBK1 as another previously unknown sensor of cellular AA levels. Thus, this proposal will also determine how AAs increase TBK1 activity to promote mTORC1 signaling and better define how the TBK1- mTOR axis promotes glucose homeostasis and insulin sensitivity in specific physiological contexts, i.e., in the obese but not lean state. This research will provide conceptual advance, as it will shift how we think about AMPK and TBK1 in metabolic control and their relationship to mTORC1. Molecular understanding of mTOR network regulation and how mTOR engages in crosstalk with other signaling systems will provide critical insights that will advance our understanding of mTOR in health and disease, which may identify new therapeutic targets for treatment of mTOR-linked disorders.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Our laboratory focuses on the structure and function of human membrane cytochrome P450 enzymes using a set of integrated structural and biochemical techniques. These monooxygenases play important roles in either the metabolism of drugs or as drug targets for prevalent diseases such as cancers, diabetes, and cardiovascular conditions, as well as rare diseases such as neuromuscular spastic paraplegia and Bietti's Crystalline Dystrophy. Many drug-metabolizing and steroidogenic P450s have been well characterized functionally and structurally by our lab, supporting improved drug design, but many other P450 drug targets are very poorly characterized. In some diseases inappropriate P450 action is implicated and inhibitors are likely to be an advantageous treatment, but the active site knowledge to design them is unknown until we determine structures. In other cases P450 mutations cause a disease, but not knowing whether the defect is in protein stability or some aspect of catalytic function similarly impedes effective progress toward developing effective therapeutics. We have a 20- year track record defining P450 interactions with ligands for various drug-metabolizing and steroidogenic human enzymes. For most we generated the first structures by X-ray crystallography. The overall goal of the proposed research for the next five years is to generate initial X-ray structures and corresponding biochemical information of a selected set of validated disease-related P450 enzymes to enable viable pursuit of drug design A subset of poorly-characterized human P450 enzymes with significant evidence supporting their roles in one or more disease states were identified and will be pursued using an overall strategy including recombinant expression and purification, identification of new ligands as necessary, evaluation of clinical mutations when appropriate, followed by integrated determination of substrate and inhibitor binding affinities, substrate metabolism, inhibitor efficacy, and active site topology, primarily by X-ray crystallography and/or pharmacophore generation. These P450/ligand interaction studies, along with proposed studies of P450 interactions with their catalytic partner proteins and membranes, are expected to provide critical foundational knowledge facilitating broad impacts across a number of major and rare diseases. The feasibility of this work rests not only on the strong premise for the selected P450 enzymes, but the investigator track record, laboratory expertise, and access to all of the required instrumentation and resources in an exceedingly strong research environment. With this combination of resources we have previously accomplished similar feats for a number of other human membrane P450 enzymes. Our overall vision is to creatively employ multiple techniques to answer some of the most critical questions about how this diverse enzyme superfamily functions and can be modulated to improve human health.