University Of Colorado

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT HIV-associated neurocognitive disorder (HAND) remains a significant challenge for individuals living with HIV, even in the era of long-term antiretroviral therapy (ART). Despite ART’s success in suppressing peripheral viral replication, viral reservoirs in the brain contribute to neuroinflammation and cognitive decline. Amyloid beta (Aβ), a peptide traditionally associated with Alzheimer’s disease, has been identified as an antiviral immune effector, capable of directly binding to viral glycoproteins and inhibiting the entry of pathogens such as HSV-1 and influenza into host cells. However, Aβ’s dual role—both neuroprotective and neurotoxic—has not been fully explored in the context of HIV infection in the brain. This project aims to investigate how Aβ influences HIV neuropathogenesis by modulating viral entry in the CNS and its effects on neuroinflammation. Specifically, we hypothesize that Aβ binds to HIV’s envelope glycoprotein (Env) to prevent viral entry into microglial cells, the primary target in the CNS, while also promoting neuroinflammation and neuronal damage through its aggregation into neurotoxic forms. To test this hypothesis, we will integrate both computational and experimental approaches. Computationally, we will use molecular docking and molecular dynamics (MD) simulations to model interactions between Aβ and HIV Env across its various states (closed and open) and in the presence of CD4 and co-receptors CCR5 or CXCR4. The simulations will also account for viral tropism (R5-, X4-, and dual-tropic strains) to understand how Aβ affects HIV's ability to infect cells based on co-receptor usage. Ultimately, these simulations will provide insights into whether Aβ stabilizes non-fusogenic conformations of Env or disrupts co-receptor engagement, thus impairing HIV infectivity. Experimentally, we will use iPSC-derived microglia to validate the computational findings, assessing the impact of Aβ on HIV infectivity, co-receptor engagement, and microglial activation through flow cytometry, qPCR, and immunofluorescence microscopy. These studies will shed light on Aβ’s dual role in HIV neuropathogenesis and identify key molecular mechanisms driving HAND progression. The outcomes of this research will provide critical insights into the mechanisms underlying the evolution of HAND and uncover novel strategies for understanding and modulating HIV-induced neuroinflammation. Ultimately, this work may contribute to improved therapeutic approaches for mitigating the neurological complications of HIV infection, particularly in the context of ART-induced viral suppression.

NIH Research Projects · FY 2026 · 2025-08

PROJECT SUMMARY Research documents concerning rates of group-related violence among sexual minority (LGB) individuals. Further, group-related violence is associated with risk for development of a host of deleterious health outcomes, including posttraumatic stress disorder (PTSD), depression, anxiety, suicidality, and a number of physical health issues (e.g., poor perceived health, gastrointestinal complaints). To date, however, no intervention work has focused on preventing these deleterious health outcomes among LGB group-related violence survivors. We propose to refine a promising brief telehealth intervention for interpersonal trauma survivors (Community Enhancement and Recovery [CARE]) to be inclusive of the experiences of LGB group-related violence survivors and then evaluate its efficacy in preventing deleterious health outcomes (e.g., PTSD, depression) among LGB recent group-related violence survivors. CARE is a two-session intervention for violence survivors and their supporters (e.g., friends, family) that seeks to facilitate natural recovery by normalizing trauma reactions, encouraging survivors to utilize discussion of their trauma reactions with a trusted person as a coping strategy, discussing ways that supporters can initiate support conversations, and assisting supporters in providing helpful disclosure reactions to survivors. CARE also includes a workbook for survivors and supporters to facilitate helpful discussions of the survivor’s trauma reactions and to reinforce intervention skills. During the Adaptation and Planning Phase, we will refine the content of CARE for inclusivity of the experiences of LGB group-related violence survivors via ongoing feedback from a Community Advisory Board of LGB community advocates, individuals involved in advocacy for survivors of violence, and LGB group-related violence survivors involved in advocacy (Aim 1a) as well as conduct an open pilot trial of the CARE with 20 survivor-supporter dyads to further refine the CARE content and evaluate the feasibility and acceptability of research procedures and program implementation (Aim 1b). During the Evaluation phase, we will via a randomized controlled trial of LGB recent group-related violence survivors and their supporters (60 survivor-supporter dyads assigned to CARE and 60 survivor-supporter dyads to a waitlist), assess the acceptability and feasibility of CARE (Aim 2a) via program observations, post-session surveys (n = 120), and exit interviews (n = 20) with survivors and supporters. We will test CARE’s efficacy in improving survivor (e.g., PTSD, depression) and supporter (e.g., secondary trauma symptoms) outcomes (Aim 2b) via surveys completed at baseline, immediate post-test, and 6-month follow-up as well as a diagnostic interview. We also will evaluate mechanisms of change of CARE (e.g., supporter disclosure reactions, survivor coping self-efficacy) via evaluation of daily ecological momentary assessment (EMA) data collected from survivors and supporters during the intervention (Aim2c). We will recruit LGB recent group-related violence survivors from across the U.S. via social media advertisements, from LGB organizations, and from survivor advocacy offices.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Precision oncology has led to a growing population of adults with advanced cancer living increasingly longer lives in the face of profound uncertainty about the future, with over half reporting moderate to high fear of cancer progression (FoP)—anxiety about the high risk or certainty of disease progression. These fears are associated with anxiety and depression, over-use of healthcare, physical symptom burden, higher treatment regret, fatigue, and, in many studies, poorer quality of life. Moreover, FoP is strongly correlated with cancer-related trauma symptoms—physical hyperarousal, intrusiveness of cancer-related thoughts/images, and avoidance of cancer- related thoughts and feelings—which are associated with similarly poor outcomes, suggesting overlapping symptoms and etiology. While behavioral interventions exist to target fear of recurrence in early-stage cancer survivors, there is a notable dearth of behavioral interventions to address FoP or cancer-related trauma symptoms in adults with advanced cancer, and no known published randomized trials of such interventions in the United States. This represents a critical gap in knowledge for the growing community of advanced cancer survivors. In addition, cutting-edge developments for the treatment of trauma in general populations have not been adapted to cancer populations. To address these gaps, we adapted a cutting-edge behavioral treatment for trauma to reduce FoP and cancer-related trauma symptoms among adults with advanced cancer. The intervention, titled EASE, is based on written exposure therapy, a highly efficacious approach for reducing trauma symptoms in general populations that is better accepted and far briefer than other gold-standard approaches. EASE adapts this effective approach to help advanced cancer patients with elevated FoP and cancer-related trauma symptoms to reduce their fear of the future by using written exposure focused on their future worst-case scenario with cancer. Informed by the NIH stage model, we evaluated EASE delivered by telehealth in an open pilot trial for 29 adults with late-stage cancer and elevated FoP and cancer-related trauma symptoms. Pilot findings show strong acceptability, feasibility, and efficacy potential. We now propose to conduct the first randomized trial of EASE, and, thus, first known randomized trial in the United States of a behavioral intervention for FoP and cancer-related trauma symptoms among adults with advanced cancer. Across both community and academic cancer clinics, this 2-arm trial (N=250) will compare EASE delivered by telehealth with Usual Care (UC), with 1:1 randomization. We aim to compare EASE to UC on FoP and cancer-related trauma symptoms (primary outcomes) and anxiety, depression, hopelessness, and quality of life, at post-intervention (Aim 1) and follow-up (Aim 2). We also will evaluate theorized mechanisms for EASE relative to UC (Aim 3). Offering EASE in both English and Spanish, and by telehealth, increases access. Simple content increases scalability. Rigorous evaluation of EASE has the potential to provide a paradigm-shifting intervention ready for dissemination and a solid empirical foundation to inform evidence-based care guidelines for distressed adults with advanced cancer.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Dysregulation of zinc homeostasis can lead to either zinc deficiency or zinc excess, resulting in a variety of human pathologies. Therefore, strict regulation of zinc trafficking and storage are essential for cellular function and human health. Robust studies of zinc biology can be conducted with a variety of model systems, and I propose to combine the genetic and experimental power of Caenorhabditis elegans with the medical relevance of human cell culture to develop a detailed and useful understanding of zinc biology. Studies from my early postdoctoral training have uncovered that lysosome-related organelles, called gut granules in C. elegans, are restructured in a zinc dependent manner; I identified an expansion compartment that increases in volume in zinc excess and deficient conditions. In addition, the low zinc homeostasis pathway appears to be conserved in C. elegans and humans, since the Low Zinc Activation enhancer element has been identified in the promoters of zinc transporters that function the low zinc response in both organisms. To build upon these preliminary results, I propose to take a multidisciplinary approach to understand how low zinc homeostasis is regulated and how lysosomes are restructured. In Aim 1, I propose to characterize the zinc-dependent expansion compartment and the membrane architecture of intestinal gut granules in C. elegans. In Aim 2, I will elucidate the regulation of zinc trafficking and morphological restructuring of human lysosomes. In Aim 3, I will characterize the regulation of the low zinc pathway in C. elegans and human cells. This proposal will capitalize on my experience with interdisciplinary techniques from cell biology, chemistry, and X-Ray physics, and expand upon them to complete my toolkit for probing zinc in biology with genetics and biochemistry. Furthermore, I will expand into human cells as a model system. The training will also build upon my extensive experience in science advocacy and equip me to be a powerful advocate for historically marginalized groups as a faculty member. My training in the K99 phase will integrate the expertise from my mentor Dr. Kornfeld and co-mentor Dr. Diwan to complete my preparation for the R00 independent phase. Training during the K99 phase will integrate the experience from my mentors, collaborators, and advisory board members to springboard my career as a scientist and science activist. My long-term career goal is to lead a team of diverse trainees performing cutting edge techniques to probe critical questions in zinc homeostasis and trafficking. I have a strong record demonstrating my abilities as a scientist, and therefore my potential as a primary investigator. I am committed to conducting leading edge science AND promoting institutional change to promote diversity and inclusion in academia. The K99/R00 award will maximize my chances of being able to achieve my goals by providing critical resources and connections that would be otherwise be lacking in my training.

NIH Research Projects · FY 2025 · 2025-07

Biology has become a data-driven, quantitative science, leading to an enormous demand for deeply trained scientists at the interface of biology and computing who engage in interdisciplinary collaboration. The Predoctoral Training Program in Biological Data Science (BDS) at the University of Colorado Boulder BioFrontiers Institute is a student-centered, interdisciplinary training program aimed at generating scientists prepared for both the challenges of big data and productive careers in biological data science. Training faculty are an outstanding, internationally recognized group of 25 researchers from five departments across two colleges, who leverage data science approaches towards research in the biomedical sciences. The program will train students in core principles of data science, including data management, analysis, visualization, as well as effective communication and collaboration, with a particular focus on rigor and reproducibility in big data research and usage. We request support for five positions in year one, 10 positions in subsequent years. Promising trainees will be selected at the end of their first year for support in years 2-3. BDS program elements include cross-department coursework, data science skill building, workshops, supergroup meetings, and seminars that collectively engage students. Coursework on responsible conduct of research and a rigor and reproducibility workshop in data science will instill students with the expectations and skillset for conducting data science research with high ethical standards. A network of academic, social, financial, and mental health resources will be in place to support interdisciplinary trainees. Trainee mentoring through peers and academic (co-)advising, formal industry partnerships and mentorship, thesis committees, and interdisciplinary collaborations also support trainee progress. The training program fosters a research group that would otherwise not have an environment to access guided training in comprehensive biological data science. BDS incorporates academia and industry perspectives to guide curriculum, assist students in building meaningful scientific networks, and enable successful career development.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Down’s Syndrome (T21) is the most common aneuploidy disorder in humans. T21 pregnancies are at an increased risk of adverse pregnancy outcomes including stillbirth and intrauterine growth restriction, resulting from placental insufficiency. T21 is also accompanied by comorbidities including congenital heart disease, which are also linked to inadequate placentation. Coincidentally, T21 placental syncytiotrophoblasts, which act as the primary maternal-fetal interface, fail to adequately fuse into multinucleated syncytial plaques needed for function. We hypothesize that defects in T21 syncytiotrophoblast differentiation occur during peri-implantation development due to dysregulation of gene expression associated with an additional copy of chromosome 21. Ultimately, these defects likely contribute to a wide range of diseases in individuals with T21. The long-term goals of our research are to provide a mechanism by which syncytiotrophoblast differentiation is disrupted in T21. Our proposed work is innovative because it combines cutting edge trophoblast differentiation protocols with functional genomics approaches. In Aim 1, we will identify transcriptional differences of T21 implantation-stage trophoblasts by differentiating six pairs of T21 and matching euploid induced pluripotent stem (iPS) cell lines into syncytiotrophoblast-like cells. For this purpose, we will use a differentiation protocol that includes BMP4, A83-01, and PD173074 (BAP). We will then characterize gene expression in these cell lines using RNA-sequencing and test the functional role of differentially expressed genes in trophoblast differentiation using knockdown and overexpression approaches. In Aim 2, we will take a hypothesis-driven approach to assess the role of NRIP1, a transcriptional co-repressor found on chromosome 21, in trophoblast differentiation. Notably, NRIP1 binds to estrogen receptor to suppress canonical estrogen-driven transcriptional responses. Our preliminary data demonstrate that attenuating estrogen signaling disrupts trophoblast differentiation in a similar manner to T21 cell lines. To better understand the role of NRIP1 in the context of Down syndrome and trophoblast differentiation, we will knockout the third copy of NRIP1 in T21 cell lines using CRISPR/Cas9 technology. We will then measure syncytiotrophoblast function through fusion indices, gene expression, and hormone secretion to determine whether decreasing NRIP1 in T21 lines rescues phenotypes observed in Down syndrome. We will also overexpress a third copy of NRIP1 in euploid lines and again measure estrogen mediated transcriptional activity and syncytiotrophoblast cell fusion to test whether increased expression of NRIP1 is sufficient to induce T21 phenotypes. These results will clarify the involvement of NRIP1 and canonical estrogen signaling in the T21 syncytiotrophoblast disease phenotype. Collectively, this work is significant because it will provide a mechanistic link between placental defects and T21 pregnancies, which may inform efforts to mitigate adverse pregnancy outcomes and comorbidities associated with Down syndrome.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT The lack of a noninvasive, cost-effective, and highly accessible technology for monitoring cerebral metabolic rate of oxygen (CMRO2) poses a critical gap in neurology and brain health. Current methods, including positron emission tomography, functional magnetic resonance imaging, diffuse optical tomography, and catheter-based jugular venous oxygen saturation monitoring, have limitations such as radiation exposure, invasiveness, limited spatial and temporal resolution, and reduced accessibility. This project aims to address this unmet need by developing the next-generation photoacoustic computed tomography through an ergodic relay (PACTER) device for noninvasive CMRO2 monitoring. In Aim 1, the device will be designed and fabricated to enable high-resolution vascular imaging of oxygen metabolism. This will involve integrating an acoustic impedance-matching layer, using longer-wavelength illumination for improved spatial resolution and penetration depth for imaging the carotid artery and internal jugular vein, and employing dual-wavelength illumination for quantifying total hemoglobin concentration and oxygen saturation. Additionally, blood flow will be measured using photoacoustic vector tomography. The device will be customized for handheld operation, ensuring easy access to imaging regions. In Aim 2, the next-generation PACTER device will be characterized and validated for monitoring CMRO2 in humans. Imaging performance, accuracy, and sensitivity will be assessed using tissue-simulating phantoms, animal models, healthy volunteers, and patients with neurological conditions. By measuring relevant parameters in the carotid artery and internal jugular vein, including blood volume, hemoglobin concentration, oxygen saturation, and blood flow, the device will enable the calculation of oxygen extraction and CMRO2. Comparisons with existing methods, such as fMRI, will be conducted, setting the stage for future clinical validation studies. The proposed research addresses the significant gap in noninvasive CMRO2 monitoring and offers transformative advancements. The development of the next-generation PACTER device will facilitate personalized diagnostics and care for neurology, neurosurgery, and critical care patients. It will provide timely and crucial information for accurate diagnosis, treatment guidance, and surgical decision-making, ultimately leading to improved medical outcomes and quality of life. Furthermore, the cost-effectiveness and accessibility of PACTER will contribute to reducing the overall cost of medical care and benefit researchers in the field. The project's innovation lies in the creation of a highly accessible device with label-free, non-ionizing, and high-spatiotemporal-resolution imaging capabilities, overcoming the limitations of current CMRO2 monitoring techniques. In summary, the next- generation PACTER device offers a novel approach to noninvasive CMRO2 monitoring, addressing a critical gap in the field. This research will facilitate progress towards significant improvements in brain health assessment, personalized diagnostics, and intraoperative monitoring, ultimately advancing our understanding of brain function and improving patient outcomes.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY/ABSTRACT Eukaryotic ubiquitination involves the conjugation of one protein to another through the sequential activity of three enzymes, E1, E2, and E3. These enzymes catalyze the transfer of ubiquitin or ubiquitin-like proteins onto target protein substrates. Ubiquitination contributes to the regulation of a wide variety of eukaryotic cellular processes, including protein degradation, gene transcription, DNA repair, and cell cycle control. Another important role for ubiquitin or ubiquitin-like protein conjugation is in the human innate antiviral immune response. During infection, the innate immune system serves as the first line of defense against viral threats. By recognizing the foreign, or “non-self”, components of the virus, the immune system activates various pathways to prevent widespread infection. Two examples of antiviral innate immune pathways that are known to be regulated by canonical ubiquitin or ubiquitin-like protein conjugation are cGAS-STING and ISG15, but many of the mechanistic details of these pathways have yet to be uncovered. Recently, operons in bacteria have been discovered that encode E1 and/or E2 ubiquitin transferase domains, and, in some instances, canonical ubiquitin-like proteins. These operons have been defined as bacterial innate immune systems that function through protein conjugation mechanisms and are evolutionarily related to the human cGAS-STING and ISG15 immune pathways. Here, I have proposed a multidisciplinary strategy to understand the molecular mechanisms of protein conjugation used by bacterial defense systems. In one of these systems, an E1-E2 ubiquitin transferase enzyme, Cap2, conjugates a non-ubiquitin-like protein, a signaling enzyme called bacterial cGAS, onto an unknown target molecule to protect bacteria from phage infection. I will use mass spectrometry-based proteomics, mechanistic biochemistry, and phage infection assays to explore how phage defense is achieved through conjugation of a non-ubiquitin-like protein (cGAS) in this system. I will also explore the mechanisms of the bacterial ISG15-like (Bil) and two other ubiquitin protein-containing defense systems using mechanistic biochemistry and mass spectrometry. Specifically, I will determine how the conjugation of canonical ubiquitin-like proteins confers defense against phage infection. Deciphering the details of ubiquitination-like processes in bacterial innate immunity will inform our understanding of the underlying mechanisms of conserved human innate immunity pathways, including cGAS-STING and ISG15. Gaining new insights into these mechanisms could pave the way for developing novel therapeutics that modulate the human immune response during viral infection.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY The opioid epidemic remains a critical public health crisis, marked by high rates of addiction, overdose, and mortality. Opioid addiction is a chronic, relapsing disorder with withdrawal often triggering anxiety and avoidance, leading to subsequent drug-seeking and taking behavior. This proposal aims to investigate the neural mechanisms underlying opioid withdrawal-induced anxiety using a combination of behavioral animal models and advanced neurotechnological techniques. Our experimental approach involves a model in which mice self- administer oral oxycodone and display somatic withdrawal symptoms when oxycodone availability is removed. My preliminary data show that mice will self-administer oral oxycodone and display withdrawal signs when oxycodone availability is removed. Further, exposure to an innately threatening visual looming stimulus prompts mice to engage in defensive behaviors, including avoidance. However, my recent work indicates that repeated exposures to this looming stimulus leads to decreased avoidance behaviors, demonstrating that these defensive behaviors are adaptive. Integrating these two models, my preliminary data show that mice undergoing opioid forced abstinence exhibit impaired adaptive defensive responses, as evidenced by their persistent avoidance behaviors toward the looming stimulus. The midbrain-striatal pathway utilizes dopamine and glutamate for learning of rewarding and threatening stimuli and reinforces avoidance behaviors. The midbrain region, substantial nigra pars lateralis (SNl) sends dopaminergic and glutamatergic projections to the tail of the striatum (TS). Preliminary data indicate that dopamine and glutamate respond to threatening stimuli. Additionally, dopamine release in the tail of the striatum (TS) initially responds to the visual looming stimulus and attenuates with repeated exposures. During opioid abstinence, dopamine and glutamate response to threatening stimuli is exaggerated, therefore, I hypothesize in opioid-abstinence disrupts dopamine and glutamate functioning in the TS, leading to sustained avoidance behavior to the looming stimulus. A combination of fiber photometry, optogenetics, and behavioral analyses of oxycodone intake and defensive behaviors will be used to target and investigate the SNl-TS pathway. In Aim 1, we will evaluate opioid withdrawal-induced changes in dopamine and glutamate release from SNl neurons to the TS. In Aim 2, we will manipulate dopamine, glutamate, and co- transmission of dopamine-glutamate from the SNl to the TS during VLS presentations and identify changes in defensive behaviors. The data generated from this project may identify novel mechanisms of neurotransmitter transmissions that may reduce opioid withdrawal-induced anxiety.

NIH Research Projects · FY 2025 · 2025-05

A. Project Summary We request funds to purchase an Evident Scientific (Olympus) SpinSR, super-resolution spinning disk confocal microscope. This spinning disk microscope will replace and update our now obsolete technology, and will also provide a gentle, high-speed super-resolution method for live and fixed samples that our current structured illumination microscopy platform (end-of-life January 2024) cannot provide. The SpinSR will be housed within the Porter Biosciences Light Microscopy Core Facility (LMCF), located at the heart of the University of Colorado Boulder main campus. Here we identify 12 Major Users and 3 Minor Users, from 4 departments, who contribute to this proposal and will use the SpinSR. This User Group represents 18 active NIH awards (9 RO1, 5 R35, 1 DP, 1 R21 and 1 PO1,) and 1 HHMI award. In 2015, we assembled a spinning disk microscope around a repurposed Yokogawa CSU-10A Nipkow disk unit, which was built in August 2002. In 2017 we purchased a demonstration unit Yokogawa CV1000 spinning disk microscope that was built in 2011 and uses a Yokogawa CSU-X1 disk unit. We’ve maintained these microscopes, and they have been essential for our Users, having supported at least 39 peer-reviewed publications and numerous grants, doctoral and undergraduate honors theses, most from NIH-funded laboratories. As these microscopes are becoming dysfunctional (detailed in Rationale section) and are severely limited by today’s standards, our Users are increasingly unable to conduct their experiments, and they cannot even pursue certain experiments due to limitations of technology. Compared to our current spinning disk confocal microscopes, the SpinSR is more efficient, with a larger FOV and faster performance, uses 6 lasers, has 2 cameras for simultaneous 2-wavelength imaging, performs gentle, high frame-rate, real-time super-resolution, has expanded range high-speed optical sectioning, multipoint photo-bleaching/photo-stimulation and superior optical performance. The science represented in our User Group is diverse, ranging from mechanisms of proteasome quality control to – pair bonding in adolescents to – neural tube biology – illustrating our need for a flexible and robustly configured spinning disk microscope. Our current spinning disk and super-resolution capabilities cannot be serviced or updated, the microscopes and computers depend on unsupported software and operating systems and cannot fulfill our current and future needs, severely limiting our users. The SpinSR will address all the shortcomings of our current systems and enable our Users to extend their research by offering new capabilities and technologies, helping them to be more productive and make new discoveries, leading to high-impact papers and successful NIH grant proposals.

NIH Research Projects · FY 2025 · 2025-05

Project Summary/Abstract We propose that the 2025 Vestibular Oriented Research Meeting will be held on the University of Colorado Anschutz Medical Campus in Aurora/Denver, Colorado on June 23-26. The overall goal of this meeting is to enhance collaborative efforts and drive better understanding of the function of the vestibular system. This meeting will bring together a diverse range of vestibular experts from around the world – including audiologists, basic scientists, neuroengineers, neurologists, neuroscientists, otologists, occupational therapists, and physical therapists. These experts will come from the communities of government and academic scientists. Building upon the successful meetings in 2019, 2021 (virtual), and 2023, the focus of the 2025 meeting will be on all aspects of vestibular function, including but not limited to (a) mechanisms and anatomy of the vestibular periphery, (b) information processing, (c) visual-vestibular integration, (d) behavioral research, including vestibulo-ocular reflexes and balance, (e) aging, and (f) vestibular systems modeling and neuroengineering. We intend to continue following the 2025 meeting, with a meeting every other year (e.g., 2027, 2029, etc). Objectives of our meeting series are to (i) promote a sense of community (ii) foster scientific discussions and interactions, (iii) support young investigators and participant diversity, and (iv) encourage collaborative research (e.g., sharing of unique resources like some of those at the Naval Medical Research Unit in Dayton). Successful attainment of our objectives will greatly enhance the pace, direction, and completion of rigorous vestibular-centric research, the discoveries of which could lead to seminal evidence necessary to move the field forward. The meetings will offer trainees (students and post-doctoral fellows) funding to offset conference attendance and participation-related costs, providing an opportunity to discuss their work, develop new ideas, and network not only with other junior investigators, but also with senior scientists, providing an unique collegial environment for professional growth. The 2025 meeting is co-organized by researchers from the University of Colorado-Boulder (PI Dr. T. Clark), the University of Colorado-Anschutz (Drs. K. Rennie and J. Hebert), and the Ohio State University (Dr. D. Merfeld). In addition, as in prior meetings, we will have a diverse group of 6-8 planning committee members. The Anschutz Medical Campus provides an outstanding co-located scientific and clinical setting to encourage the intense invigorating discussions planned for this collegial gathering, in addition to providing a distinctive opportunity to visit world-class vestibular research laboratories and clinical facilities.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY We request funds to purchase a high-resolution and high-sensitivity liquid chromatography-mass spectrometry system for the analysis of small molecules and biomolecules at the University of Colorado Boulder (CU-Boulder) as well as other research institutions in Colorado. The proposed instrument consists of a Waters SELECT SERIES Cyclic Ion Mobility QTOF (SS-cIMS) mass spectrometer, a Waters ACQUITY Premier UPLC, and a Waters ACQUITY HDX Manager UPLC. It will be housed in the Central Analytical Laboratory (CAL), which is the mass spectrometry service core facility at CU-Boulder. It will be used to support mass spectrometry needs for measuring small molecule high-resolution accurate mass, biomolecule mass including intact protein mass analysis, peptide LC-MS/MS and mapping of post-translational modifications, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This new instrument is needed to replace an existing Waters Synapt G2 system that is now 15 years old and in poor condition. Importantly, Waters Corp. will soon end service support for the Synapt G2 on December 31, 2024. Therefore, we have an urgent need to replace the Synapt G2 with a newer QTOF instrument that can fill the critical needs in synthetic chemistry and biomolecular analysis that is prevalent across our research community. In addition, the proposed instrument would enable the CAL facility to expand its research capabilities, by enabling new experiments using ion mobility mass spectrometry and native mass spectrometry to evaluate chemical and conformational heterogeneity of large macromolecular complexes. These will synergize and support emerging research among our large community of structural biology researchers at CU-Boulder, who are using single particle cryo-electron microscopy and cryo-electron tomography to determine structures of large complexes at atomic resolution.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Neural tube defects (NTDs) occur in ~1-2 in 1000 pregnancies globally every year and constitute one of the top three most common congenital developmental differences in the world. An embryo with an NTD develops with an area of partially enclosed or completely exposed central nervous tissue, which can lead to complications both during pregnancy and after birth. Some NTDs are so severe they lead to lethality, and even milder forms of NTDs lead to a decreased quality of life. NTDs can have many negative consequences for the people they affect, and improving identification of pregnancies that are at risk for an NTD would benefit many people around the world. One known NTD risk factor is maternal diabetes, which exposes the embryo to elevated levels of glucose. The relevance of glucose to NTDs is one of the factors that makes Insulin Receptor Substrate 1 (IRS-1) an attractive candidate for investigation as a potential player in NTD risk. In addition to the role of IRS-1 in mediating glucose uptake, glucose utilization, and cell growth, multiple patients with NTDs have mutations in IRS1 (collaboration with the Gleeson Lab, UCSD). These pieces of evidence focus my proposed project on IRS-1 and its intersections with maternal diabetes and embryonic glucose response. Much work has been done on glucose from the maternal perspective, but little is known about glucose homeostasis or the role of IRS-1 in the embryo. This study aims to use IRS-1 to clarify the relationship between NTD risk and the embryonic response to environmental glucose. By understanding how perturbing IRS-1 levels can dysregulate glucose homeostasis and lead to NTDs, we can improve identification of pregnancies at risk for NTDs. To achieve these goals, the proposed study has two aims. Specific Aim 1 will investigate the role of IRS-1 in cellular glucose consumption dynamics using mouse neural progenitor cell lines and primary cells with a genetic knock-out of the Irs1 gene. Specific Aim 2 will examine IRS-1 loss using an established Irs1 knockout mouse line. Embryos from this model will be cultured and live-imaged ex utero to dynamically assess the effects of IRS-1 loss on the morphology of the neural tube as it closes. To understand potential interactions between maternal diabetes and Irs1 loss, female mice from this line will also be treated with streptozotocin to induce diabetes, and NTD incidence in their offspring will be measured. All experiments will be conducted in the Niswander lab, which is part of the Molecular, Cellular, and Developmental Biology Department at the University of Colorado Boulder. Both the lab and department provide excellent resources including a supportive environment, quality equipment, and a wealth of knowledge from experienced scientists. These resources will support my journey towards my career goal of becoming an independent research scientist in academia, as well as my intermediary training goals of accumulating technical skills in developmental biology, improving my science communication skills, and mentoring future scientists.

NIH Research Projects · FY 2026 · 2025-02

Mechanisms of Lipid A Modification Impacting Antimicrobial Resistance Marcelo Sousa University of Colorado Boulder Project Summary Human Cationic AntiMicrobial Peptides (CAMPs) represent a conserved branch of the innate immune system. CAMPs constitute a first line of defense against bacterial colonization that is particularly important in exposed tissues and surfaces such as the skin, eyes, airways and lungs. Furthermore, CAMP antibiotics such as colistin and related polymyxins are a class of essential drugs in clinical use to treat recalcitrant infection with multidrug resistant Gram-negative bacteria. These include the “urgent threat” pathogens carbapenem resistant Acinetobacter baumannii, Carbapenem Resistant Enterobacteriaceae (CRE) as well as “serious threat” pathogens such as Multidrug Resistant (MDR) Pseudomonas aeruginosa. However, these and other Gram- negative pathogens have evolved mechanisms to modify lipid A in the outer membrane of bacteria (the CAMP/colistin target) most commonly with 4-amino-arabinose (Ara4N), which result in resistance to the bactericidal activity of CAMPs and colistin. This is a significant clinical problem with pathogens that produce persistent infections with high mortality rates. Therefore, development of inhibitors of the Ara4N-Lipid A modification pathway is a desirable therapeutic strategy that would result in potentiators of colistin against deadly multidrug resistant pathogens. The enzymes responsible for the biosynthesis of Ara4N-Lipid A are clustered in the ArnBCADTEF operon. Mutation of any of these genes abolishes Ara4N addition to Lipid A and colistin resistance. Therefore, these proteins are validated targets for drug development to abolish colistin resistance. However, the biochemical characterization of several of these targets is either lacking or incomplete. Furthermore, in vitro activity assays are either currently unavailable or inadequate for quantitative evaluation of putative inhibitors. This knowledge gap impairs development of colistin adjuvants that could improve the efficacy of this life-saving antibiotic. We will close this gap with an integrated, collaborative research program to structurally and functionally characterize three proteins directly responsible for CAMP/colistin resistance, screen for probes to define inhibitory strategies, and develop a platform to quantitative test impact of target activity in vitro, in bacteria and in a tissue culture Salmonella infection model. Successful completion of this multidisciplinary program will result in new insights into the mechanisms of lipid A modification leading to CAMP/colistin resistance and provide fully characterized targets with validated assays and inhibitory probes. This represents a superb platform for future development of adjuvant drugs that augment the efficacy of colistin in treatment of infections with multidrug resistant bacteria.

NIH Research Projects · FY 2026 · 2025-02

Summary The objective of this project is to investigate groundbreaking approaches for in-vivo high-resolution imaging of fine corneal structures with confocal-like sectioning capability, wide field-of-view (FOV), and polarization and phase sensitive detection. With further development, the proposed technique should be easy to implement and use in a clinical environment while overcoming the limitations of current techniques. Despite remarkable developments and advances in imaging technologies, in particular, by the advent of optical coherence tomography (OCT) that has transformed the diagnosis and management of ophthalmic diseases, the clinical assessment of cellular structures of the cornea remains challenging in patients, while visualization of sub- cellular structures remains beyond reach. While clinical OCT provides a lateral resolution of about 15µm, in-vivo confocal microscopy (IVCM) can achieve much higher (~1µm) lateral resolution. Several issues have prevented the widespread adoption of IVCM for routine cornea imaging in clinical practice, e.g., requiring physical contact with the patient’s cornea and capturing a small FOV. Corneal disorders are among the leading causes of blindness worldwide. Therefore, new methods are needed to better resolve corneal structures in the living human eye in a reliable and consistent fashion. The project will investigate speckle illumination holographic imaging with the additional capability to record the polarization of the optical fields, thus providing information about the anisotropy of the cornea. It will also address the capture of three-dimensional information over the depth and width of the cornea. After a system is developed and optimized, a proof-of-concept validation will be carried out in vivo in healthy adult human volunteers. The proposed imaging approach will facilitate the evaluation of corneal cell and nerve morphology in healthy and pre/post-operative individuals, thus having a substantial impact in the study, prevention, diagnosis, treatment, and monitoring of various corneal diseases. With the cornea serving as a privileged site for the direct visualization and study of numerous general physiologic and pathologic processes, the significance of the results however extends well beyond ophthalmology.

NIH Research Projects · FY 2026 · 2025-01

Project Summary This is an application in response to FOA number, PAR-24-022 (NIBIB), “Trailblazer Award for New and Early Stage Investigators (R21 Clinical Trial Optional)”. Recently, we demonstrated non-mechanical scanning for imaging. This technology can be used for a miniature forward-viewing endoscopic micro-optical coherence tomography (µOCT) system to image tissue during surgery. The endoscope will enable great insight into human illnesses such as cardiovascular disease, cancer, and neurological disorders. While µOCT is a mature technology, miniaturization of a system into a probe has faced challenges. Galvos are large and bulky, MEMs mirrors (both electrostatic and electrothermal) require non-negligible powers, mechanically moving parts, and are limited in speed. In contrast, our endoscopic probe relies on a compact, non-mechanical electrowetting adaptive optical component that shows great promise for imaging applications. We will first validate our device by designing and demonstrating a low-voltage component operating at kHz speeds. Next, we will develop a miniaturized µOCT endoscope system with an integrated electrowetting prism scanner. We will demonstrate both systems on phantom tissue. The results can be used to extend the technology to imaging the cardiovascular system. AIM 1: TECHNICAL APPROACH Demonstrate kHz spectral-domain µOCT using a low voltage electrowetting prism scanner AIM 2: TECHNICAL APPROACH Demonstrate a miniaturized µOCT endoscope system with an integrated electrowetting prism scanner

NIH Research Projects · FY 2026 · 2024-12

Down’s Syndrome (T21) is the most common aneuploidy disorder in humans. T21 pregnancies are at an increased risk of adverse pregnancy outcomes including stillbirth and intrauterine growth restriction resulting from placental insufficiency. T21 is also accompanied by comorbidities including congenital heart disease, also linked to inadequate placentation. Coincidentally, T21 placental syncytiotrophoblasts that act as the primary barriers at the maternal-fetal interface fail to adequately fuse into multinucleated syncytial plaques, and the underlying etiology is unknown. We hypothesize that defects in T21 syncytiotrophoblast differentiation occur during peri-implantation development and contribute to a wide range of diseases in individuals with T21. The long-term goals of our research are to provide a mechanism by which syncytiotrophoblast differentiation is disrupted in T21 and provide opportunities for therapeutic intervention. In Aim 1, we will identify transcriptional differences of T21 implantation-stage trophoblasts by differentiating six pairs of T21 and matching euploid induced pluripotent stem (iPS) cell lines into syncytiotrophoblast-like cells with treatment of BMP4, A83-01, and PD173074 (BAP) and performing RNA-sequencing. In Aim 2, we will assess canonical estrogen-driven transcription in T21 and euploid BAP-treated iPS cells. We will knockdown the third copy of NRIP1, an estrogen receptor inhibitor on chromosome 21, with CRISPR/Cas9 technology in T21 BAP-treated iPS cells to determine the influence of estrogen receptor inhibition on T21 syncytiotrophoblast fusion. We will also overexpress a third copy of NRIP1 in euploid lines and measure estrogen mediated transcriptional activity and syncytiotrophoblast cell fusion. These results will help to confirm or deny the involvement of NRIP1 and canonical estrogen signaling in the T21 syncytiotrophoblast disease phenotype. In Aim 3, we will differentiate T21 and euploid iPSCs to cardiomyocytes and supplement them with conditioned medium from T21 and euploid trophoblast cultures to reveal a relationship between impaired T21 trophoblasts on cardiomyocyte development. In summary, our proposal uses cutting edge models and techniques that have not been used in T21 trophoblast research to demonstrate the significance of abnormal placentation on long-term health.

NIH Research Projects · FY 2026 · 2024-11

Modifications of Lipid A with Phospho-Ethanolamine Impacting Polymyxin Resistance Marcelo Sousa University of Colorado Boulder Project Summary Human Cationic AntiMicrobial Peptides (CAMPs) represent a conserved branch of the innate immune system. CAMPs constitute a first line of defense against bacterial colonization that is particularly important in exposed tissues and surfaces such as the skin, eyes, airways and lungs. Furthermore, CAMP antibiotics such as colistin and related polymyxins are a class of essential drugs in clinical use to treat recalcitrant infection with multidrug resistant Gram-negative bacteria. These include the “urgent threat” pathogens carbapenem resistant Acinetobacter baumannii, Carbapenem Resistant Enterobacteriaceae (CRE) as well as “serious threat” pathogens such as Multidrug Resistant (MDR) Pseudomonas aeruginosa. However, these and other Gram- negative pathogens have acquired mechanisms to attach cationic modifiers such as phosphoethanolamine (pEtN) to lipid A in the outer membrane of bacteria (the CAMP/colistin target) which result in resistance to the bactericidal activity of CAMPs and colistin. This is a significant clinical problem with pathogens that produce persistent infections with high mortality rates. Therefore, development of inhibitors of the enzyme that catalyzes the Lipid A modification with phosphoethanolamine (pEtN) is a desirable therapeutic strategy that would result in potentiators of colistin against deadly multidrug resistant pathogens. A pEtN transferase is required for pEtN- lipid A biosynthesis and colistin resistance. Therefore, this protein is validated target for drug development to combat colistin resistance. However, the biochemical characterization of pEtN transferase is incomplete. Furthermore, in vitro activity assays are either currently unavailable or inadequate for quantitative evaluation of putative inhibitors. This knowledge gap impairs development of colistin adjuvants that could improve the efficacy of this life-saving antibiotic. We will close this gap with an integrated, collaborative research program to structurally and functionally characterize pEtN transferases directly responsible for CAMP/colistin resistance, screen for probes to define inhibitory strategies, and develop a platform to quantitative test impact of target activity in vitro. Successful completion of this exploratory multidisciplinary program will result in new insights into the mechanisms of lipid A modification leading to CAMP/colistin resistance and provide a fully characterized target with validated assays and active site binding probes. This represents a superb platform for future development of adjuvant drugs that augment the efficacy of colistin in treatment of infections with multidrug resistant bacteria.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Subsidies for contraception and even the right to use contraception are often justified on the basis of their benefits to women’s socioeconomic lives. While the benefits of expanding contraceptive access have been documented, reductions in access are now under consideration. Because reductions in access operate differently than expansions, they will not simply reverse the effects of expansions. Therefore, it is critical to understand how such reductions impact women’s social and economic outcomes. This project uses newly-developed data and one of the earliest and largest reductions in access to contraception to generate the first estimates of reduced contraceptive access in the US. The project will estimate the causal impact of reduced access to contraception using a massive policy experiment in Texas. In 2011, Texas decreased its grant-based contraceptive funding program by 2/3 and deprioritized dedicated family planning providers. These actions caused clinic closures, reduced access to the most effective methods, and were associated with increases in teen fertility. In partnership with the U.S. Census Bureau, the project will (Aim 1A) expand an individual-level longitudinal dataset, Reproduction in People’s Lives (RIPL), recording women’s fertility and human capital formation for nearly all reproductive-age U.S. women 2000-2026. RIPL links restricted individual-level microdata for the entire United States from the Census Numident, Master Address File–Auxiliary Reference File, three decennial Censuses, individual IRS tax filings, the Census Household Composition Key, and the 2005-2026 American Community Survey. The project will also (Aim 1B) integrate original survey-based and administrative clinic- level data on the spatial intensity of Texas reductions in contraceptive access into RIPL. And importantly, the project will (Aim 1C) yield a modular version of RIPL, so researchers on Census Bureau-approved projects can use our documented code to construct components of RIPL for replication, extension, and their own use. Changes in life course outcomes as Texans experienced reductions in access contraception will be compared to changes in comparison states using a difference-in-differences approach. In this way the project will (Aim 2) assess the impact of reductions in access to contraception on women’s fertility over their life course. Similarly, the project will (Aim 3) assess the impact of reductions in access to contraception on women’s economic outcomes and human capital accumulation. For each outcome, whether impacts vary by age at exposure, family of origin socioeconomic status, and race/ethnicity, as well as whether fertility mediates the impact of reduced access (Aim 4) on women’s socioeconomic lives will be assessed. Project findings will inform ongoing debates over the causal effects of access to contraception and fertility timing on socioeconomic outcomes. The data and methods developed may also be used by researchers on other approved projects to investigate the life course in ways previously prevented by data limitations.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Older adults are particularly vulnerable to health impacts from climate-related events such as heat waves, hurricanes, wildfires, and floods. These impacts include both physical and mental health such as experiencing higher overall rates of mortality, likelihood of injury, disruption of medical treatments, and the experience of Post-Traumatic Stress Disorder. Understanding these disproportionate impacts is particularly important given projections of a climate future with more frequent and more intense environmental extremes. Yet progress in research on the Aging-Climate-Health nexus has been slowed by disciplinary silos and data challenges. The proposed virtual Center on Aging, Climate, and Health (CACHE) responds directly to these challenges by taking a multi-pronged approach to the development of infrastructure to accelerate research advancements. The development of CACHE will take place in two phases with Years 1-2 (R61) involving several demonstration research projects integrating social and environmental data and the development of a strong online presence to facilitate sharing of data, code, and training materials in support of interdisciplinary scholarship. While a large amount of long-term climate and disaster data are publicly available, they are in formats unfamiliar to health and social scientists and, thus, require technical and substantive expertise to use. CACHE will offer the necessary training and resources to overcome these substantial barriers. During this initial phase, the Center will also recruit an interdisciplinary affiliate base through outreach efforts involving its Executive Committee and Advisory Board that represents environmental demography, aging, health, and climate science. A proof-of-concept training session on climate data will be offered prior to the end of Year 2. In Years 3-5 (R33), we build on this foundational work by expanding to offer additional trainings on multi-scale social-environmental analysis, data visualization, and other important methodological topics, while also further developing the Center’s blog to publicly grapple with research challenges in this arena. CACHE will also offer seed grants to support the early stages of promising research and, in support of building an interdisciplinary community, will provide funding for topical workshops to bring together scholars interested in critical areas with knowledge gaps. Yearly mini conferences offer a space to share ideas, provide feedback, and identify important questions and collaborative opportunities. In all, the work emphasizes the bridging of data platforms and the crossing of interdisciplinary silos to bring multiple disciplinary perspectives to bear within the increasingly important topical area of Aging, Climate, and Health.

NIH Research Projects · FY 2025 · 2024-09

SUMMARY/ABSTRACT For many years, scientists have been trying to find new therapeutic drugs. However, small molecules are still imperfect tools for influencing human physiology. The chemical space that our known drugs occupy is extremely small, representing a tiny sliver of the estimated 10^63 possible drug-like molecules. What’s more, the targets of these drugs make up just about 3.5% of the roughly 20,000 proteins in the human proteome. This means almost 97% of the proteome is still untouched for drug intervention. These two factors suggest there’s immense potential for discovering novel chemical structures to combat disease. However, systematically exploring such enormous chemical spaces is physically unachievable. To synthesize even milligram scale representations of this chemical space would result in a mass surpassing that of the known universe. This practical obstacle partly explains why only a small portion of the proteins in the proteome have been pharmacologically targeted. To overcome this diversity enumeration hurdle, we aim to introduce a completely new class of molecules capable of spontaneously “shape shifting” at physiological temperature. This unique ability will allow them to assume a wide array of rapidly interchanging structural isomers, essentially behaving like many molecules in one. We plan to demonstrate this technology through the development of synthetic bullvalene amino acids that can be seamlessly incorporated into existing methodologies for combinatorial solid phase peptide synthesis to create shape shifting cyclic peptides (SSCP). Our ultimate goal is to create a dynamic and adaptable library that far outpaces any known library in terms of diversity. We aim to utilize this technology to identify single molecular entities that display multi- target pharmacological properties.

NIH Research Projects · FY 2025 · 2024-09

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. In conventional metabolic signaling, secreted protein hormones such as insulin and glucagon elicit metabolic responses in target cells by binding to surface receptors. Recently, a new paradigm of metabolic signaling has emerged, where RNAs act as signaling molecules. For effective intercellular signaling, RNAs (e.g., microRNAs) must be transferred from donor to recipient cells via extracellular vesicles (EVs)—membranous particles released by nearly all cell types. EVs are released from donor cells either through secretion of intraluminal vesicles enclosed in multivesicular bodies/endosomes (exosomes) or by budding from the plasma membrane (microvesicles). When EVs fuse with the surface or endosome of recipient cells, the encapsulated RNAs are delivered into the recipient cell's cytosol, where they regulate gene expression. Previous studies have primarily provided evidence of EV-dependent RNA signaling through the deletion of RNAs from EVs and the inhibition of EV release from donor cells. However, these studies have not demonstrated the critical role of EV fusion with recipient cells, leaving open the possibility that metabolic responses might be triggered by non-RNA molecules co-purified with EVs or present on the EV surface. In this pilot project, we will conduct biochemical and genetic experiments to identify proteins crucial for EV-mediated delivery of adipose microRNAs into liver cells, with a focus on noncanonical proteins that have been overlooked in previous research. If such proteins are identified, we will develop genetic strategies to test whether EV fusion-mediated RNA delivery is necessary for metabolic signaling between adipocytes and liver cells. These pilot studies are expected to provide insights into the pathogenesis of metabolic diseases associated with RNA signaling and facilitate the development of novel therapeutics.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Spinal cord injury (SCI) results in lifelong sensorimotor deficits, leading to chronic mobility impairments and loss of functional independence. The restoration of walking remains a highly valued goal for persons with chronic, incomplete SCI (iSCI) reducing sequalae due to immobility. Modest breathing modest bouts of low oxygen (acute intermittent hypoxia; AIH), is shown to enhance motor recovery in both spinally injured rats and persons with SCI by inducing adaptive reorganization in spared neural circuitry. Detailed mechanistic studies of AIH-induced respiratory and non-respiratory motor plasticity in rats provide a fundamental basis to advance our understanding and optimize the clinical benefits of AIH therapy. However, the underlying neural mechanisms that contribute to improvements in functional recovery in humans with SCI remain unclear. Importantly, we do not currently do not fully understand why AIH augments walking performance in humans with iSCI. In previous studies, approximately 33% of study participants with chronic iSCI did not respond to the AIH intervention, not achieving clinically meaningful improvements in motor performance. In order to optimize the clinical translation of AIH as an adjuvant for walking rehabilitation, we will examine two critical knowledge gaps: (1) a lack of understanding of the underlying mechanisms of AIH induced plasticity, (2) the identification of predictive biomarkers to determine who optimally responds to AIH. To address the first knowledge gap, we will validate the degree to which AIH engages the same cellular mechanisms in humans as observed in spinally injured rodents by utilizing enzyme linked immunosorbent assays to quantify AIH induced synthesis of serotonin and brain derived neurotrophic factor. To address the second knowledge gap, we will use magnetic resonance imaging to measure descending tract connectivity as predictive biomarkers to identify responders in AIH. Responders will be identified by quantifying gains in walking performance after the AIH intervention. To further support this approach, we will utilize transcranial magnetic stimulation to clarify the functional relevance of AIH induced changes in the excitability of descending tract connectivity. Together, these studies will not only advance the detection of patients who will optimally respond but also identify patient specific factors that facilitate or inhibit AIH induced functional recovery. The insights gained from this study will enhance our understanding of how to harness AIH-induced plasticity to promote meaningful recovery and will profoundly impact the lives of persons with chronic spinal injuries.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The mammalian cell cycle is commonly conceived as a well-understood, hardwired, invariant pathway. Emerging work, however, indicates that the cell cycle is much more plastic than generally believed, with multiple adaptive routes through the cell cycle under different conditions. This plasticity makes the cell cycle robust to environmental perturbations, but also drives adaptive drug resistance to targeted cell-cycle inhibitors. A fresh look at the dynamics and pliability of cell-cycle progression will reveal new principles that predict dependence on a particular cell-cycle node and new strategies to suppress adaptive cell-cycle rewiring. Cyclin-Dependent Kinases (CDKs) are key enzymes that drive cell proliferation, and consequently, multiple CDK inhibitors are in development to suppress unwanted cell proliferation. However, cells eventually find a way around these drugs to resume proliferation. A plausible hypothesis is that cells leverage cell-cycle plasticity to pursue alternative paths through the cell cycle. In one striking example, inhibition of CDK2 leads to rapid loss of substrate phosphorylation as expected, but then CDK2 substrate phosphorylation rebounds within several hours. This rebound depends on CDK4 and CDK6, which insulate the cell from fluctuations in CDK2 activity by maintaining Rb hyper-phosphorylation and E2F transcription. This enables CDK2 re-activation and eventual cell-cycle completion, even in the presence of potent CDK2 inhibitors. My lab has pioneered the development of a set of powerful time-lapse microscopy tools to visualize rapid drug responses in single, living cells. Here, we will apply our technology to determine the mechanisms driving this unusual rebound in CDK2 activity observed upon CDK2 inhibition. First, we will test the role of the p16 CDK4/6 inhibitor protein on the CDK2 activity rebound. Second, we will test whether CDK2-dependent degradation of Cyclin D modulates the CDK2 activity rebound. Third, we will identify additional mechanisms underlying the robustness of Rb phosphorylation to inhibition of CDK2 and show how long-term drug pressure unleashes the plasticity of CDKs. Since our findings are likely to be broadly applicable beyond CDK2 inhibitors, our proposed work will fill long-standing gaps in our understanding of how the cell cycle is wired for success in the face of perturbations.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The human body has ~37 trillion cells and over 200 different cell types. Polycomb Repressive Complex (PRC1, PRC2) and DNA methyltransferase 1 (DNMT1) are evolutionarily conserved chromatin modifier enzymes that are essential for establishing and maintaining cellular identity by silencing lineage-inappropriate genes during embryonic development and cellular differentiation. PRC1, PRC2, and DNMT1 are also key enzymes in cardiac development, and defective activity of these enzymes is a hallmark of cardiac diseases and loss of cell identity in various cancers and tumors. Polycomb-mediated gene silencing involves the coordinated activity of PRC1 and PRC2 through histone post-translational modifications. PRC1 is a histone E3 ligase that catalyzes the mono- ubiquitination of H2A at lysine 119 (H2AK119ub1) on nucleosomes, which aids in the recruitment and activation of PRC2 for the trimethylation of histone H3 (H3K27me3) leading to gene silencing. Similar crosstalk between trimethylation of histone H3 at lysine 9 (H3K9me3) and mono-ubiquitination of histone H3 at lysine 18 and 23 (H3K18K23ub2) is thought to be responsible for the recruitment and activation of DNMT1 at replication forks for the maintenance of DNA methylation. The major research goal in the lab over the next five years is to uncover the mechanistic basis for the recruitment and activation of PRC1 and DNMT1 on chromatin. We will utilize an integrated approach combining cryo-electron microscopy, chemical biology, mass spectrometry, and biochemical assays to (a) uncover the mechanistic basis for how DNA and histone post- translational modifications activate PRC1 and DNMT1 on chromatin and (b) design and develop new inhibitor and activator candidates that can target specific regions of PRC2 that are important for chromatin interactions and enzymatic activity. We will use biochemical mutational and activity assays to validate our results from the structural studies. We will also biochemically characterize how known disease-causing mutations lead to defects in the enzymatic activity of PRC1 and DNMT1 on chromatin. The vision of our research program is to provide novel structural and mechanistic insights into the role played by chromatin in activating gene-silencing enzymes such as PRC1, PRC2, and DNMT1. We envisage that such insights will aid in developing small molecule effectors targeting these enzymes in malignancies such as solid tumors, which have so far lacked targeted treatments. One example of this structural biology approach is our proposed work on developing effector molecules targeting specific regions of PRC2 in this proposal.