University Of Colorado

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract Voice disorders affect 5-10% of the United States workforce, with an even higher incidence (≈ 50%) among professional voice users such as teachers and fitness instructors. A key factor contributing to these disorders is dehydration of the vocal fold tissue, which increases stiffness and reduces pliability. As individuals speak with dehydrated folds, they are prone to vocal overuse, leading to dysphonia and potential lesions. Yet the mechanisms of vocal fold dehydration and its effects on phonation remain poorly understood, largely because the vocal folds are difficult to access directly. This project circumvents limited experimental access using novel computational approaches to test hypotheses about hydration, pitch, loudness, and fluid atomization. In Aim 1, we will develop a fluid–structure interaction model that couples exhaled airflow to poroelastic, hyperelastic vocal fold tissues. This model will simulate oscillatory dynamics and the internal flow of interstitial fluid, enabling us to study how vibration may lead to progressive dehydration. We will validate the model against prior simulations of oscillation frequencies, mucosal wave propagation, and modal behavior. In Aim 2, we will model how Faraday- like instabilities on the tissue surface can drive droplet ejection. We will extract pixel-wise surface kinematics from video laryngoscopy and use those data to simulate surface-level fluid instabilities that droplet ejection. By identifying the role of tissue motion, saturation, and geometry in driving aerosol generation, this work will reveal the physical mechanisms that affect tissue hydration and cause fluid emission from the surface. The results will inform clinical understanding and medical interventions used to diagnose and treat dysphonia.

NIH Research Projects · FY 2026 · 2026-06

Project Summary Alcohol use disorder (AUD) is debilitating, with enormous costs. Among the negative effects of alcohol misuse, disrupted sleep is among the most significant contributors of progression to AUD and relapse from recovery. Though there has been some previous research on sleep in AUD, interventions that improved sleep had little to no effect on alcohol misuse nor vice versa. Given the significant impact of these intertwined behaviors, new treatments for disrupted sleep that have low abuse potential and could aid in recovery from disordered alcohol use are critically needed. One treatment target that could be thus further explored is the endocannabinoid system (ECS), a shared biological pathway for both sleep and alcohol misuse with research suggesting cannabinoid receptors and neurotransmitters anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) are involved in the regulation of sleep as well as with the reward system. ECS activity has been of interest as a possible target for treatment of both disrupted sleep and AUD separately, but not yet explored as treatment for disturbed sleep in the context of alcohol use. Additionally, one molecule that should be explored further in this context is cannabidiol (CBD), an exogenous cannabinoid that is known to be involved in both sleep and reward systems. There is increasing evidence CBD may benefit sleep behaviors, potentially mitigating sleep disruptions related to alcohol use. While trial results are largely mixed, some encouraging findings in combination with the low abuse liability of CBD have led to calls in the literature for more work on CBD and sleep specifically in AUD. Therefore, the primary objective of this study will be to conduct a randomized placebo-controlled trial to explore effects of CBD on sleep and alcohol use behaviors in adults reporting heavy drinking, including exploratory mediational analysis of the role of sleep quality. This study is a logical progression of my research thus far in the areas of psychopharmacology, alcohol use, and substance use. The corresponding career development plan will leverage the aims of the study to develop expertise in behavioral sleep medicine, establishment and administration of clinical trials with investigational products, and advanced statistical methods in multilevel modeling and mediational analysis. The environment for the project is highly supportive, including leading experts in the requisite subject areas working out of some of the highest ranking, most collaborative institutions in the U.S. The completion of the study and attainment of the training objectives will position me to establish an independent clinical research career in a growing field with high public health significance.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY/ABSTRACT Common pathological signatures of heart failure include decreased contractility and increased stiffness, leading to decreased cardiac output. Identifying the underlying molecular mechanisms behind these phenotypes is critical for targeted therapeutic development to address heart disease. Pythons represent a unique model organism to study cardiac hypertrophy and altered cardiac function. After feeding, pythons undergo rapid physiological cardiac hypertrophy within 72 hours, followed by subsequent regression. Recent work from our lab also showed that pythons rapidly increase cardiac myofibril active tension and decrease myofibril passive tension after feeding. Identifying the regulatory mechanisms that promote rapid tension changes in the post-fed python heart may allow for the discovery of novel, conserved mechanistic targets for modifying cardiac function in humans. We conducted a preliminary global phospho-proteomics study with samples from fasted and fed ball python ventricles. I identified 19 differentially phosphorylated resides on cardiac myosin heavy chain (a key determinant of active tension/force) and 133 differentially phosphorylated sites on titin (a key determinant of passive tension). The majority of these sites are conserved in humans and reside within known myosin and titin functional domains. We have also generated biochemical data suggesting that ATP turnover of myosin is altered after feeding. The goals of this work are to further our mechanistic understanding of how phosphorylation of specific residues regulates titin stiffness and be the first study to assess how phosphorylation in the motor domain and proximal rod region affects myosin heavy chain function. Specifically, I will conduct single molecule atomic force microscopy (with the Perkins Lab at CU Boulder) and thermodynamic stability assays to determine how phospho-mimetic mutations affect the elastic and thermodynamic properties of titin. I will also use phospho-null/mimetic mutations within the motor and proximal rod regions of cardiac myosin heavy chain to mimic changes that occur after feeding and characterize the impact on myosin function and structural states with biochemical and electron microscopy assays. My long- term goals are to identify how pythons rapidly tune cardiac function and undergo hypertrophy in a physiological fashion (specifically through alternative splicing and post-translational modifications), and how this compares with mammalian models of physiological and pathological remodeling. This fellowship will allow me to learn single molecule force microscopy techniques from world-leading experts, gain expertise with myosin functional assays that our lab has significant experience in, and improve my background in muscle biophysics. These skills will help prepare me for a future career as an independent investigator in the field of cardiac remodeling.

NIH Research Projects · FY 2026 · 2026-06

Project Summary/Abstract Klocko Laboratory research overview: Eukaryotic genomes are precisely and non-stochastically organized in the nucleus for proper function, including gene expression and chromatin formation. In humans, genome disorder can cause to aberrant gene expression and uncontrolled cellular growth, leading to oncogenic tumor formation, as observed in neuroblastoma and pancreatic cancers. However, a complete understanding of the mechanisms necessary to organize eukaryotic genomes is not known in any species, which is a critical gap in our collective knowledge. Specifically, many (epi)genetic factors have not been examined for roles in organizing genomic DNA, including for how silent heterochromatin is segregated at the nuclear periphery from active euchromatin. We use the innovative fungal organism Neurospora crassa as a model for humans, given the similarities of its epigenetic regulation and extent of DNA compaction with humans. The Neurospora genome contains active euchromatin and silent heterochromatin, marked by identical histone post-translational modifications that are catalyzed by homologous proteins as humans. However, the small, genetically tractable, haploid Neurospora genome allows cost-efficient genomic studies, and its chromatin machinery is not essential, as strains harboring single deletions of chromatin modifying proteins are viable. By using Neurospora crassa as a model fungus, we can also directly understand how chromatin properties affect the genome organization of fungal pathogens of humans. My lab assesses the high-resolution genome organization of wild type fungal species, including Neurospora crassa, and studies how chromatin composition or the genome synteny changes impact genome organization and function. We are also characterizing the fundamental mechanisms underlying hetero- or euchromatin formation and the regulation of gene expression in several fungal species, focusing on Neurospora and Ogataea clade yeasts. Five-year goals: I want to complete several manuscripts for which we already have significant data collected, including the impact of translocations on Neurospora genome organization and the characterization of chromatin and genome organization in Ogataea species. I will continue to guide/mentor UCCS (under)graduate students on new experiments, and their subsequent data analysis, that assess the contribution of DNA binding proteins, telomeres, and histone deacetylases/acetyltransferases on chromatin composition and genome organization. I will have UCCS students present their research at international conferences to enhance their research training. Klocko Research Program Vision: My long-term goal is to substantially contribute to our collective knowledge for how fungal genomes are organized, including the underlying mechanisms required for genome organization, and how changes to organization alter genome function. Specifically, I want to explore the inherent principles of chromatin formation and how they contribute to gene regulation in heterochromatic and euchromatic genome regions using novel experimental directions on which I guide UCCS (under)graduate students. I strive to mentor the next generation of research scientists in preparation for biomedical research careers.

NIH Research Projects · FY 2026 · 2026-05

Project Summary Methyl CpG binding protein 2 (MeCP2) is a known regulator of brain development. Mutations in this X-linked gene cause Severe Neonatal-Onset Encephalopathy with Microcephaly in males and the neurodevelopmental disorder Rett Syndrome in females. Causal mutations occur anywhere within MeCP2, including the understudied intervening domain. The intervening domain includes two arginine-glycine (RG) repeat sequences and a lysine rich region, both RNA binding motifs that are evidence of an RNA binding function. Few studies have investigated the potential for MeCP2 to interact with RNA; thus, how MeCP2-RNA binding impacts brain development remains unknown. MeCP2 binds various RNAs, both coding and noncoding, however, the binding regions, identity of bound RNAs, and how this binding may regulate gene expression have not been well characterized. My studies focus on identifying specific RNAs bound by MeCP2 and the role of the intervening domain of MeCP2 in brain development. I created an intervening domain deletion iPSC cell line and used directed differentiation to cerebral organoids which shows profound molecular and cellular defects in the deletion line. I hypothesize that key neuronal RNAs bind to the intervening domain of MeCP2, and that this interaction is essential for proper brain development. To test this hypothesis, Aim 1 will identify RNA sequences that bind to the intervening domain of MeCP2 and quantify alterations in RNA expression. Aim 2 will characterize phenotypic outcomes of iPSC-derived human neural cells and cerebral organoids with the MeCP2 intervening domain deletion. As mutations in MeCP2 are the genetic cause of neurodevelopmental disorders and disrupted RNA regulation is linked to abnormal neural development, my studies have important implications for MeCP2-RNA binding as potential mechanisms for neurological disorders.

NIH Research Projects · FY 2026 · 2026-05

Project Summary The proposed work aims to identify the neural mechanisms of visual selection, which is heightened visual salience of task-relevant information. The visual system receives constant sensory input that must be parsed to select important information. For instance, drivers must detect road signs, cars, and pedestrians to determine the proper response. In the lab, this is studied using Rapid Serial Visual Presentation (RSVP) in which stimuli are presented at 10 Hz in one location and observers must respond to pre-defined “targets” (e.g., report letters, but not digits). Although the visual system can select one target at this rate, it fails to select a second presented shortly after. This transient deficit has been termed an “attentional blink”; however, the research is a mixture of single-task (e.g., report letters, not digits) and task-switching paradigms (e.g., report a white letter, then a black X). Indeed, task-switching variants may be due to an attentional deficit, single-task variants potentially reflect visual dynamics. In brief, the single-task second target deficit might reflect “conceptual repetition blindness”, defined as a transient inability to perceive that the second target belongs to the category of targets. I report data from two pilot experiments in which observers report either two words defined by their category (single-task) or one word representing a number and another defined by category (task-switching). Different patterns emerged which suggests the involvement of separate neural mechanisms. Prior work is mixed regarding whether N400 components track the second target deficit with a key difference being whether the tasks are switching. To properly test this hypothesis, I plan to conduct similar experiments utilizing semantic word stimuli and test whether known markers of early semantic processing (N170 ERP component) and memory updating (N400) are differently influenced based on task. More specifically, I predict that early, visual ERPs will also track the second target deficit in the single-task version but not in the task-switching version. This hypothesis will be tested across three aims that assess different kinds of visual selection. Aim 1 will examine semantic selection. Aim 2 will examine orthographic case selection (e.g., upper versus lower case), and Aim 3 will examine objects class (e.g., words versus faces). In each aim, it is predicted that the single-task version will reveal a visual ERP component that tracks the second target deficit, but the nature of the component will depend on the nature of the selection. For instance, orthographic case selection might reflect an earlier component (e.g., the P100), whereas object class (words versus faces), might reflect different hemispheres. I will use machine learning classifiers to identify EEG responses that best predict behavior and stimulus class. Finally, Aim 4 will test this theory by applying a dynamic neural network model that previously explained repetition blindness as the consequence of neural habituation. Using equivalent dipole modeling, the model will be applied to the full RSVP sequence to identify the cortical locations of the layers underlying the second target deficit.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract This proposal is aimed at developing a novel magnetic nanotransducer that can provide deep tissue electric stimulation. Electrical stimulation has long been used to treat various neurological conditions such as Parkinson’s, epilepsy, pain, or sensory deficits. But it is invasive and has limitations associated the electrodes that need to be implanted. Transcranial magnetic stimulation is a non-invasive technique but suffers from poor spatial selectivity and high power consumption. Optogenetic approaches are hampered by the limited light penetration in brain tissues and usually require an implantable device. A non-invasive technique that can deliver sufficiently strong stimulation to reliably trigger neural activity, and allow targeting of specific areas or cell types, could enable designing effective, specific and long-lasting brain implants to treat a number of neurological conditions much more effectively than what’s possible today. To address this long-standing challenge, we propose to develop a novel nanotransducer that can generate electric potentials up to ~100 mV upon application of an external magnetic field. The nanotransducers have a size of a few hundred nanometers and can be directly injected into tissues or organs. Once placed in the target area, the produced voltages are controlled remotely by an external magnetic field. Thanks to the excellent tissue penetration of magnetic fields but also to the novel design of the nanotransducer, the operating distance is on the order of 1 ~ 10 centimeters, enabling truly deep tissue stimulation. Compared to the current non-invasive technologies, our approach will provide orders of magnitude stronger electrical stimulations. In this proposal, we will develop an ingenuous fabrication method to produce the nanotransducers and validate their efficacy through a comprehensive set of in-vitro, in-vivo and behavioral experiments.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY There is a need to understand how deviations in glycosaminoglycan (GAG) content affect fiber level- mechanics so we can better inform regenerative therapies that aim to restore function to tendinopathic and aged tendons. Various studies investigating the influence of GAGs and proteoglycans (PGs) on tendon mechanics at the fiber and tissue scale have been conducted, usually via knockout of 1 – 2 components or non-specific enzymatic degradation. However, the effects of enzymatic treatments are inconclusive as most studies do not consider hyaluronic acid (HA), a linear GAG made up of repeating disaccharide units. Despite the simplicity, HA is integral in development, homeostasis and repair by regulating swelling pressure, tissue hydration and cell behavior. To test the effect of HA on fiber scale mechanics, we developed a new methodology that leverages laser ablation of collagen fibers within intact tendons. Excitingly, we observed enzymatic depletion of HA in murine extensor carpi radialis longus (ECRL) tendons significantly reduced the amount individual collagen fibers retract after laser ablation. This preliminary data led us to hypothesize strain recovery in type I collagen fibers is facilitated by interfibrillar HA. Specifically, disruption of HA and/or HA-binding partners will decrease strain recovery of type I collagen fibers. We will test the effect of GAG depletion and (re)addition on fiber-level mechanics of both energy storing (plantaris) and positional (ECRL) murine tendons, as well as in a model where GAG content is disrupted (aged mice). We will use the HA-specific Hyal from S. hyalurolyticus to target HA and compare the mechanical response when sulfated GAGs (sGAGs) are removed via chondroitinase ABC (ChABC). To confirm what components of the ECM were removed via enzymatic digestion, we will use proteomics, ELISA (HA) and dimethylmethylene blue (sGAGs) assays. Finally, we will adapt a strategy previously developed for cartilage by our collaborator, Jason Burdick, to reintroduce HA back into tendons. Comparison of the response of these tendons to Hyal and ChABC in Aim 1 will enable us to see if the effect of HA on fiber strain recovery is broadly applicable to all tendons or unique to the ECRL. To see how fiber sliding is affected in a model of native GAG disruption, we will investigate the effect of HA removal on aged tendons. Finally, we will use proteomics and biochemical assays for GAGs to identify how the ECM changes as a function of enzyme treatment and aging. To directly test if HA alone or HA binding partners affect strain recovery, we will reintroduce HA to the digested tendons in Aim 2. Furthermore, we will investigate if the addition of HA to undigested tendons increases strain recovery in control adult and aged tendons, which will inform how current HA-based treatments can affect tissue mechanics. Demonstration that exogenous HA enhances strain recovery in control and aged tendons will pave the way for new treatments that can reduce pathological stiffening and enhance functionality.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Major depressive disorder (MDD) has long been theorized to reflect an overly broad disorder class that collapses across heterogenous risk pathways. A rate limiting factor to examining the divergent validity of MDD subtypes using genomic methods is a lack of sufficiently powered data. As part of the proposed project, we will utilize Co-I Dr. Lewis’ role as a leader in depression genetics and co-chair of the PGC MDD working group to put together the largest genome-wide association study (GWAS) yet performed for various MDD subtypes, including sex-stratified, atypical, postpartum, and severe MDD. In addition, we will employ Genomic LOSEM, a novel method introduced in the grant for examining non-linear changes in genetic signal that we will use to examine how different ages at onset and socioeconomic status shift MDD genetic architecture. The subtype GWAS and Genomic LOSEM package will be made available as public resources. Standard univariate approaches that focus strictly on either meta-analyzing across MDD in all its forms or analysis of a particular subtype are unable to parse genetic risk pathways that are broadly relevant to MDD from those that are unique to a specific subtype. In addition, family-based approaches are pragmatically limited to examining a handful of subtypes at a time and cannot describe underlying biology. Genomic Structural Equation Modeling (Genomic SEM) is an innovative, multivariate framework developed by the grant PI Dr. Grotzinger for modeling genetic overlap derived from GWAS data. The well-powered GWAS of MDD subtypes will be used as input to Genomic SEM models that will formally disambiguate shared and subtype-specific genetic signal. A unique advantage of Genomic SEM is that even mutually exclusive subtypes can be included in the same statistical model. The remaining analyses will characterize subtype-specific genetic signal at varying levels of biological granularity, including estimating genetic overlap with clinically relevant external correlates (e.g., cognition, other psychiatric disorders). By applying Stratified Genomic SEM, a novel extension for estimating multivariate functional enrichment, we will characterize biological pathways involved in subtype specific risk. These biological pathways can include, for example, genes expressed early in development, in certain brain regions, or in specific types of neurons. At the gene expression level, Transcriptome-wide SEM will be used to identify the lists of genes uniquely associated with an MDD subtypes. These results will be cross-referenced with the Connectivity Map drug repurposing dataset to identify existing pharmacological interventions that may have therapeutic benefit. By utilizing sex-stratified GWAS summary statistics we will explicitly consider biological sex as a moderator of relevant genetic pathways. In addition, expanding African, East Asian, and LatinX ancestry GWAS datasets, LD-scores, functional annotations, gene expression weights and cross-ancestry methods will allow us to extend the grant aims across diverse samples. Our analyses will collectively provide the most comprehensive evaluation to-date of subtype-specific etiology within MDD.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY/ABSTRACT Proper regulation of mRNA transcription is essential for normal cellular metabolism, and mis-regulation can lead to multiple pathologies and disease states. In recent years it has become clear that transcriptional termination at the 3' ends of genes is mis-regulated in response to cellular stress and some diseases. For example, in response to a short heat shock, human RNA polymerase II (Pol II) reads through the typical 3' end of the vast majority of mRNA genes and continues transcribing for many kilobases into intergenic regions and neighboring genes. The mechanisms that control transcription termination in response to cellular stress are largely unknown. Importantly, transcriptional 3' end read-through also occurs at thousands of genes in healthy human tissues and cell types. The parameters that dictate whether a given gene exhibits read-through transcription during normal cellular metabolism are not clear. The proposed research will reveal mechanisms that control how read-through transcripts are generated in human cells both in response to heat shock, and in the non-stressed state. At its core, termination of Pol II transcription involves two steps – cleavage of the nascent RNA to generate the 3' end of the mRNA and removal of the polymerase from the genome – both of which will be studied. In Specific Aim 1 experiments will uncover mechanisms that regulate the RNA cleavage step of transcription termination in response to heat shock. Supporting data show that after heat shock the cleavage step of termination is defective at the majority of genes; however, over a thousand genes show proper cleavage during heat shock despite having extensive read-through. The proposed research will reveal the mechanisms by which 3' end mRNA cleavage is differentially controlled in a gene-specific fashion in response to heat shock. Experiments in Specific Aim 2 will determine mechanisms that regulate Pol II displacement from the genome after RNA cleavage. Preliminary experiments have identified genes in non-heat shocked and heat shocked cells where cleavage and read- through are uncoupled. These genes exhibit strong RNA cleavage, but also show extensive read-through transcription, suggesting termination at these genes is regulated via removal of Pol II from the genome. These gene sets will be used to understand what controls the displacement of Pol II after proper cleavage, with a focus on the movement of the polymerase and the activity of the exonuclease Xrn2, leveraging a combination of cell-based experiments and in vitro single molecule studies. Together the proposed studies define mechanisms controlling transcriptional termination in cells growing normally and in response to cellular stress.

NIH Research Projects · FY 2026 · 2026-04

Project Abstract This MIRA award request will unify two areas of R01-funded research in the laboratory of Deborah Wuttke at the University of Colorado Boulder. The common theme is probing the multi-layer regulatory networks that modulate gene expression and cellular homeostasis through regulation by nucleic acids. Specifically, the program will address outstanding questions regarding the non-canonical recognition of RNA by DNA-binding transcription factors (TFs) and the management of ssDNA by replication A (RPA) type factors. During the last grant period, we discovered a robust, and unexpected, RNA-binding activity inherent in numerous transcription factors. This RNA-binding activity was found to be mediated by extended versions of their DNA-binding domains and directly compete with DNA binding. Furthermore, we found that this activity was structure-specific rather than sequence specific in vitro. These observations raise critical questions regarding the role of RNA binding by TFs in transcriptional regulation and provide the direct motivation for the research program described here. The next steps are to further define and understand the specificity for the in vivo RNA targets and determine the impact TF RNA interactions has on the transcriptional program. We will take advantage of biochemically validated separation of function mutants that allow us to independently disrupt either RNA- or DNA-binding activity in cells and probe their mechanism of action using integrated transcriptomics and single molecule imaging. The impact of this program is high because understanding the roles of genome-wide pervasive transcription and how this activity influences diverse cellular processes remains a major unanswered question in biology. Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are required for chromosome stability and cellular proliferation. These structures are essential for human health because dysregulation of either telomere protection or telomerase activity causes many inherited and acquired human diseases, with telomere dysfunction closely tied to cancer and aging. Our program is focused on two protein complexes that manage ssDNA in many chromosomal contexts, including at telomeres and replication forks. Human CST is a heterotrimeric protein complex that protects and maintains sites of G-rich ssDNA throughout the genome, acting prominently at telomeres through binding the conserved G-rich overhang to coordinate the activities of telomerase and Pol-alpha primase. Mutants of CST are associated with a range of human diseases characteristic of proliferation defects. The structural, functional and biochemical parallels between the CST and RPA complexes suggest a highly tuned interplay of their activities that allow for their crosstalk in the management of difficult G-rich regions of chromatin and our recent data point to the intricate integration of the general DNA-maintenance and telomere machineries. This is key to understanding the basic biology of chromosome maintenance and the catastrophic consequences of its misregulation.

NIH Research Projects · FY 2026 · 2026-03

Project Summary Our DNA contains many enigmatic elements called transposons, which are genetic parasites that make up over half of our genetic material. In healthy cells, these elements are usually silenced, but they can become active in cancer cells. In our study, we explore a unique process called "exonization," where transposons can sometimes become incorporated into the messenger RNAs that contain cellular instructions for making proteins. This process creates altered versions, or isoforms, of key proteins. Specifically, we discovered that one of these changes affects the IFNAR2 gene, which produces a receptor that is essential for interferon (IFN) signaling. The interferon (IFN) signaling pathway is a critical pathway in both antiviral and anti-tumor immunity. However, many cancers have evolved ways to block this protective mechanism, making treatments like chemotherapy and immunotherapy less effective. Our findings show that an exonized version of IFNAR2 acts as a “decoy,” preventing the immune system from responding to critical interferon signals. We propose that dysregulation of this version makes cancer cells more resistant to treatments that rely on intact IFN signaling, such as immunotherapies and certain chemotherapy drugs. We aim to study whether improper regulation of this novel version of the gene in cancer may be a hidden contributor to immune dysregulation, effectively dampening the body’s immune response and allowing the tumor to evade destruction. To address this, our project has three main goals: First, we will investigate how this altered version of IFNAR2 impacts the effectiveness of cancer therapies. By manipulating cancer cells to change the levels of the normal and altered IFNAR2 isoforms, we will test how this affects their sensitivity to treatment, which could lead to new strategies for overcoming treatment resistance in cancer. Second, we will explore the underlying causes of this faulty gene splicing. Using advanced genetic tools, we will identify the proteins and regulatory factors that control this process in cancer cells. By pinpointing the exact mechanisms driving the production of the decoy receptor, we can identify potential drug targets that might reverse this immune evasion strategy. Finally, we will expand our focus to search for other instances where transposons alter immune genes in cancer. By analyzing tumor samples at the single-cell level, we aim to identify new exonized versions of immune genes that contribute to immune evasion. This broader search could uncover additional targets for future cancer therapies. By understanding these hidden alterations in immune signaling, our work may eventually improve outcomes for patients who currently experience poor responses to immunotherapies and other treatments that rely on a functional immune response. This study could ultimately lead to the development of novel drugs that make the immune system to fight cancer more effectively.

NIH Research Projects · FY 2026 · 2026-03

Project Summary Mood disorders in adolescence are prevalent, disabling, and associated with future chronicity and severity of mood problems in adulthood, making this a developmental period in which effective early intervention is especially important. A critical common ingredient of many interventions for adolescent mood disorders is inter- session monitoring of behaviors and mood; such monitoring is used by patients and practitioners to support case conceptualization, identify patient-specific maladaptive behaviors, and evaluate treatment outcomes. However, adherence to inter-session monitoring is low among adolescents, undermining intervention effectiveness. To optimize and support delivery of effective interventions for adolescent mood disorders, we are in need of novel, low-burden strategies for monitoring behavior and mood. The proposed study aims to address this urgent need by validating and translating a novel smartphone-based intervention monitoring approach (Sensor-Based Intervention Monitoring, SBIM). Aims will be tested in an adolescent sample (n=33, ages 13-19 years) engaged in interventions for mood disorders through the Helen and Arthur E. Johnson Depression Center or the Child and Family Clinic at the University of Colorado Anschutz Medical Campus. Study participants will complete a baseline evaluation followed by an eight-week period of SBIM using smartphones to collect native digital sensors (e.g., GPS, accelerometer, log data of screen use, app use, call/text activity, and ambient light/sound) and ecological momentary assessment (EMA) of mood symptoms. Daily behaviors are measured by extracting behavioral features from digital sensors, and include sensor-based measures of behavioral activation, impulsivity, physical activity, social withdrawal, sleep/circadian disturbances, and goal-directedness. SBIM Reports, providing inter- session monitoring information about patient-specific behaviors, mood, and behavioral predictors of mood, are delivered to clinicians and patients biweekly. Clinicians and patients report on acceptability, appropriateness, and feasibility of SBIM, and treatment outcomes, biweekly. Aim 1 centers on rigorously validating SBIM, testing the predictive accuracy of personalized models and comparing personalized to general models. Aim 2 evaluates feasibility, appropriateness, and acceptability of SBIM for both patients and clinical providers, and Aim 3 tests convergent validity of SBIM with standard treatment outcomes. Exploratory Aim 4 explores subgroups to compare differences in SBIM outcomes between interventions (behavioral, medication management), diagnoses (unipolar, bipolar) and as a function of sex, age, or pubertal stage. Ultimately, we aim that this work will develop and validate a novel monitoring tool and explore translation to the clinic, providing a foundation for research that scales and investigates SBIM outcomes.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY / ABSTRACT The long-term goal of this project is to transform women’s health by investigating the novel biological matrix of menstrual blood for measurement of environmental chemical exposure, advancing understanding of endocrine disrupting chemicals (EDCs) and reproductive system disorders. One such condition, endometriosis, is estrogen- driven, affects ~10% of people with uteruses, and can confer chronic, debilitating symptoms, including pelvic pain, menstrual pain, and heavy menstrual bleeding. EDCs may affect endometriosis through alteration of estrogen pathways, but studies have yielded inconsistent results. Unlike venous blood, menstrual blood contains endometrial tissue, allowing for the characterization of environmental chemical exposure in the target organ of the endometrium. A paucity of data exists on the chemicals present in menstrual blood, as well as sources of exposure (such as menstrual product use) and connection to reproductive health. To address this gap, this prospective study will measure EDCs in menstrual blood, including metals and perfluoroalkyl substances (PFAS), to which the US general population is ubiquitously exposed. The study will recruit 225 menstruating university students aged 18-49 in the Boulder, CO metro area (the ‘Menses Study’). This population will be followed for 3 menstrual cycles. Menstrual blood will be collected in cycles 2 and 3 using an established, successful collection protocol. A venous blood sample will also be collected during cycle 2, corresponding with menstrual blood collection. Daily menstrual blood samples will be collected in cycle 3. Data on menstrual product use and menstrual cycle characteristics, particularly symptoms of menstrual pain and heavy menstrual bleeding which are commonly reported with reproductive system disorders, will also be collected. The specific aims are: (Aim 1) Characterize exogenous chemical exposures in menstrual blood: (1a) Use standard targeted methods to quantify concentrations of 17 metals (As, Ba, Ca, Cd, Co, Cr, Cu, Hg, Pb, Mn, Ni, Sb, Se, Sr, Tl, V, and Zn) and 24 PFAS, (1b) Use a discovery-driven approach to elucidate novel exogenous compounds (non-targeted chemical analysis), (1c) Characterize variability of targeted and non-targeted chemicals within and between menstrual periods, (1d) Compare chemical concentrations in menstrual blood to those in venous blood collected concurrently. (Aim 2) Evaluate sources of exposure unique to menstruators, including use of tampons and other menstrual products, in relation to metals, PFAS and non-targeted compounds in menstrual blood. (Aim 3) Determine associations between metals, PFAS, non-targeted compounds and menstrual symptoms indicative of reproductive system disorders: menstrual bleeding severity and menstrual pain. This project aligns with the NIH crosscutting theme of advancing research on women’s health by kickstarting exposure assessment science in an understudied matrix indicative of uterine health: menstrual blood.

NIH Research Projects · FY 2026 · 2026-02

PROJECT SUMMARY Many metabolic responses involve the trafficking of proteins within the endomembrane system. A major branch of membrane protein trafficking is vesicle fusion, which entails the merging of membrane-enclosed vesicles with their target membranes. Vesicle fusion mediates key metabolic processes, including the translocation of the glucose transporter GLUT4, insulin secretion, glucagon release, and GLP-1 secretion. Imbalances in these vesicle fusion processes are associated with metabolic disorders such as insulin resistance (IR) and type 2 diabetes (T2D). To devise effective therapeutic strategies for these disorders, it is crucial to understand how protein-protein networks mediate and regulate vesicle fusion in these metabolic pathways. Our research focuses on the vesicle fusion pathway critical for the insulin-dependent translocation of GLUT4 in adipocytes and muscle cells. In our preliminary studies, we developed CRISPR-based genetic platforms and identified new players in the vesicle fusion pathway. Additionally, through biochemical and biophysical assays, we uncovered novel protein-protein and protein-membrane binding modes in GLUT4 vesicle fusion. In this work, we aim to expand these preliminary findings to elucidate how the soluble and membrane proteins act in concert to mediate and regulate the vesicle fusion reaction and to understand how fusion kinetics are affected by changes in lipid composition. Our genetic experiments employ cultured adipose and muscle cells differentiated from progenitor cells, as well as genetically modified mouse strains. We will also use a novel platform of mature human adipocytes derived from differentiation of human pluripotent stem cells. As a crucial step towards grasping the molecular basis of IR, we will use mouse models to explore how a high-fat diet-induced IR condition affects vesicle fusion mediators. The experiments proposed here are expected to provide key mechanistic insights into the molecular basis of metabolic vesicle fusion, and will pave the way for understanding IR and T2D. Ultimately, these findings will likely contribute to the development of new treatments for these metabolic disorders.

NIH Research Projects · FY 2026 · 2026-01

PROJECT ABSTRACT The proposed research will assess the impact adolescent friendship networks have on the psychosocial health of immigrant youth (i.e., the 1st and 2nd generation). Immigrant youth are a vulnerable population, with disproportionately high prevalence of adverse psychosocial health outcomes, including higher rates of mental health disorders, and lower sense of belonging, self-esteem, and wellbeing. Adolescence is a critical developmental stage, marked by the ascension in the complexity and importance of peer friendships. Although immigrant youth’s friendship networks look different from non-immigrant youth’s networks in a variety of ways, very little research have examined whether and how these friendship patterns and processes may explain the immigrant-based disparities in psychosocial health. This proposed research will bring more contemporary data to the forefront of research on immigrant youth’s school friendships, generate new and rare data on immigrant youth’s non-school friendships, and use advanced social network analytical methods to provide a more comprehensive examination of the impacts adolescent friendship networks have on the psychosocial health of immigrant youth. During the K99 phase, Dr. Khuu will focus on friendships developed in school, a major peer context shared by both immigrant and nonimmigrant youth. AIM 1 is to identify and understand differences in friendship patterns and processes between immigrant and nonimmigrant youth. AIM 2 is to compare measures of psychosocial health between these two groups and test whether differential friendship patterns and processes explain differences in psychosocial health. During the R00 phase, Dr. Khuu will leverage her training in survey design and network sampling methods as well as in adolescent development and psychosocial health to lead a new data collection effort on immigrant youth’s friendship networks extending beyond school. AIM 3 is to understand how the social contexts of friendships shape friendship patterns and composition. AIM 4 is to test the relationship between immigrant youth’s psychosocial health and these friendship measures. As a sub aim, Dr. Khuu will also take the opportunity to examine heterogeneity among immigrant youth, focusing particularly on the distinctions between refugee and non-refugee youth. Dr. Khuu’s career goal is to become a leading research authority on the friendships, health, and critical life outcomes of immigrant youth. The training and findings of the proposed research will position her favorably to pursue an R01 grant, enabling her to propose a more expansive, longitudinal study that explores the social integration and health of immigrant youth, with a specific focus on refugee youth, who have resettled in a diversity of new immigrant destinations in the United States.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY CANDIDATE: Kevin O. Murray, PhD, is a postdoctoral fellow training in vascular aging at the University of Colorado (CU) Boulder. In this K01 application, Dr. Murray aims to determine the efficacy of mitochondrial- targeted antioxidant (MitoQ) treatment for improving cerebrovascular function in estrogen-deficient postmenopausal (PME-) women. His immediate goal is to acquire the research training and professional skills necessary to transition to an independent, extramurally funded investigator. His long-term goal is to establish his own research program with a focus on identifying novel, evidence-based alternative interventions that improve vascular function and prevent or delay the development of age- and menopause-related comorbidities and chronic diseases. CAREER DEVELOPMENT PLAN: Dr. Murray’s career development plan consists of: 1) determining the efficacy of MitoQ vs placebo for improving cerebrovascular function in PME- women; 2) acquiring new skills to assess cerebrovascular function to support his proposed research plan; 3) professional skill development through coursework and attendance/presentations at weekly journal clubs, CU seminars, and national scientific meetings; and (4) regular interactions with his mentor team. ENVIRONMENT: The environment for Dr. Murray’s training plan will be exceptional. Dr. Murray’s primary mentor, Dr. Douglas Seals, and co-mentor, Dr. Kerrie Moreau, are internationally recognized, NIH-funded scientists with strong records of successful mentoring in translational biomedical research. Co-mentor Dr. Philip Ainslie is the Canada Research Chair in Cerebrovascular Physiology with extensive experience assessing cerebrovascular function. Consulting mentors: (1) Dr. Michel Chonchol has served as physician of record for the previous and ongoing MitoQ trials conducted by the primary mentor’s laboratory and will provide additional mentoring related to performing clinical research; (2) Dr. Zhying You is a Senior Biostatistician in the Department of Medicine at the CU Anschutz Medical Campus and provides mentoring to trainees and faculty conducting clinical trials; (3) Dr. Zachary Clayton is an expert in assessing vascular function in mice, particularly through the role of changes in the circulating milieu; and (4) Dr. Aurelie Ledreux is an expert in brain aging and the role of extracellular vesicles. RESEARCH: PME- women are at an increased risk of cardiovascular disease (CVD), including stroke, due, in part, to cerebrovascular dysfunction in response to excess mitochondrial reactive oxygen species and consequent reductions in nitric oxide bioavailability and increases in arterial stiffness. This clinical trial will determine whether an oral supplement called MitoQ, which reduces the production of damaging reactive oxygen species from mitochondria, can improve cerebrovascular function in PME- women, and will provide insight into the biological reasons (mechanisms) by which supplementation with MitoQ exerts these benefits. Overall, this research will provide scientific evidence supporting the use of MitoQ for improving cerebrovascular function and decreasing CVD risk, such as stroke, in PME- women.

NIH Research Projects · FY 2025 · 2025-09

The objective of this Pathway to Independence Award is to support Dr. Chen in establishing an independent research career investigating developmental pathways to alcohol use/misuse in adolescence and early adulthood. The overarching goal of this project is to understand how and why parental factors are significantly associated with adolescent early alcohol initiation. Understanding mechanisms behind such associations is crucial for developing effective prevention programs that target specific parental behaviors to prevent alcohol-related harms. Specifically, the proposed research integrates two important genetically informed approaches (family-based genetic designs and genomics) to examine three potential causes of associations between parental drinking behaviors/psychopathology and adolescent early alcohol initiation: (1) parents and children share genes; (2) parents and children live in the same environmental contexts; and (3) parental factors directly influence their children’s alcohol initiation. Specific Aim 1 (the K99 phase) uses family-based genetic designs that include twins, adoptees, and their extended family members; Specific Aim 2 (the R00 phase) integrates genomic data into models developed for family-based genetic studies to examine parent-child associations using data from an NIH-supported program ECHO including 50k+ families. Specific Aim 2 further examines potential mediators and moderators of parental influences to help understand how a prevention program on adolescent alcohol use can best target parental behavior, and who will benefit the most from such a program. Dr. Chen will be supported by a highly experienced mentoring team with expertise in genetics, alcohol use, and developmental psychology, as well as rich resources offered at the Institute for Behavioral Genetics in relation to genetic methods and the etiology of youth alcohol use. Specific training objectives include: (1) gaining proficiency in multilevel influences on youth alcohol use; (2) enhancing expertise in advanced family-based genetic designs; (3) integrating genomic data into advanced family-based genetic models; and (4) acquiring professional skills to facilitate the transition into an independent research career. These training objectives will be achieved through meetings with mentors, directed readings, hands-on experiences in methodological skills, and working on specific research projects. Dr. Chen will also complete courses and attend seminars/workshops offered at the Institute for Behavioral Genetics and beyond to further her knowledge in genetics and youth alcohol use. By targeting multilevel factors including parents, peers, neighborhood environments, and broader social contexts, the research proposed in this project has the potential to help inform effective prevention programs that aim to delay drinking onset and prevent alcohol-related harms.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY This project examines early-life mortality trends in the United States (US) between 1990 and 2024. Findings from the project will provide the most detailed and comprehensive insight into the ways that changes in cause-specific death rates have reshaped early-life survival chances for American birth cohorts, and how these survival chances contrast with experiences in peer countries. The project will use official US mortality data at county, state, and national levels; national-level data for other high-income countries; and several US datasets containing county and states' demographic, economic, and sociopolitical indicators to examine how early-life US mortality trends have differed by sex, age, race/ethnicity, and across subnational regional contexts. In Specific Aim 1, we will estimate how causes of death have contributed to male and female US mortality trends in infancy (age 0), childhood (ages 1-14), adolescence/transition to adulthood (ages 15-24), and young adulthood (ages 25-44). We will also decompose US-peer differences in life expectancy by cause of death at these ages, thereby documenting how causes of death in early life have contributed to the widening US-peer longevity gaps in four time periods: 1990–1999, 2000–2009, 2010–2019, and 2019–2024. Through Specific Aim 2 we will broaden our investigations in Aim 1 to examine heterogeneity in these early-life trends by US state, sex, age, race/ethnicity, and SES levels across US county-groups. Critically, in both Aim 1 and Aim 2, we will examine US early-life mortality trends in a comparative and international context so that we produce knowledge about both absolute changes (e.g., how cause-specific death rates have changed over time for a specific US population) as well as relative changes (e.g., how US-peer differences in cause-specific death rates have changed over time). Thus, by contrasting US mortality trends against those experienced by populations in other high-income peer countries, the project will provide key insights into the characteristics, nature, and size of early-life mortality inequalities within the United States. In Specific Aim 3, we will estimate cohort differences in cause-specific death rates in early-life and calculate cohort-based changes in survival chances to midlife (i.e., to age 25, age 35, and age 45). Cohort analysis can actually reveal larger mortality changes – and larger mortality inequalities in these changes – than estimates derived from period-based analyses alone. Finally, in Specific Aim 4, We will estimate how changes in early-life cause-specific death rates in the United States and peer countries vary across birth cohorts and by time periods, providing key insights into the trends' causes. By examining early-life mortality trends in the United States through both (i) a cohort framework, and (ii) within an international context, this project will significantly improve our understanding of recent changes in cause-specific mortality among young US populations, and how these changes contribute to the US longevity disadvantage as well as contribute to widening mortality inequalities within the United States.

NIH Research Projects · FY 2025 · 2025-09

Neural tube closure (NTC) is the embryonic process that forms the precursor to our brain and spinal cord. Neural progenitor cells (NPCs) are the predominant cell type in the neural tube and are exquisitely regulated to balance cell specification, proliferation, and differentiation—all processes critical for NTC. When NPCs and NTC are disrupted by genetic and environmental insults, the neural tube does not close and results in a neural tube defect (NTD) such as Spina Bifida and Anencephaly. Metabolic disorders that dysregulate lipid homeostasis (maternal diabetes and obesity) are risk factors for NTDs in humans. In mouse, several metabolic and genetic NTDs models with dysregulated lipid homeostasis can be prevented by vitamin E supplementation. The correlation of disrupted lipid homeostasis to vitamin E rescue points to a critical question, can we predict if a genetic model is likely vitamin E responsive and therefore help prevent NTDs? In order to predict if vitamin E is a suitable strategy, we must figure out the key targets of vitamin E, and to narrow the scope of targets, we must understand what happens in NPCs when lipid homeostasis is dysregulated. Lipids are required for signaling pathways used in NPC specification, membrane composition which enable cell shape changes during proliferation and differentiation, and energetically fueling and maintaining cell health enabling the aforementioned processes. Although we know that lipid homeostasis is in principle important for NTC and NPC function, our understanding of how lipids are regulated in NPCs and their discrete functions in these cells is severely understudied. This proposal takes an integrated approach to address the relationship between lipids, NPC fate, and NTC and to evaluate vitamin E responsive processes using the FAF2 knockout mouse model as a test piece. FAF2 is an ER membrane protein that regulates lipid synthesis and lipid droplet maintenance, thus enabling investigation of lipid homeostasis from two distinct angles. The primary goal of my mentored phase is to use cutting-edge imaging, - omics, and genetic manipulation to identify the consequences of disrupted lipid synthesis and lipid droplet maintenance on NTC and NPC regulation downstream of FAF2 knockout in embryos and NPCs. These results will prepare me to independently adapt this experimental framework to assess the interplay between lipid homeostasis and vitamin E. Overall this work is of high significance to our understanding of structural birth defects and will provide insight into what makes lipid dysregulation so dangerous to neural tube closure. While in my K99 phase, I will take advantage of the rich academic environment of CU Boulder and its relationship with nearby CU Anschutz to adapt novel techniques to study lipid biology in embryos and develop robust analyses. My long-term career goal is to establish an independent research program that uses cell and developmental biology techniques to investigate gene-environment interactions in lipid-related NTDs. The techniques and methodologies developed in this proposal will create a scaffold of strategies by which any other lipid-associated gene or pathway can be interrogated for its role in NTC and NPC regulation in my independent lab.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Ribonucleoprotein (RNP) granules are membraneless organelles formed from RNA and RNA binding proteins (RBPs) through biomolecular condensation or liquid-liquid phase separation. Stress granules (SGs) are prototypical RNP granules, which form transiently under stresses such as arsenite exposure, UV damage, and viral infection. The unifying thread between these stresses is translation shutdown due to eiF2a phosphorylation, leading to rapid increases in the concentration of lowly bound cytoplasmic RNA, which coalesces with RBPs to form SGs. Chronic stress exposures or mutations in SG RBPs lead to constitutive SGs that behave more like aggregates than condensates. These stable SGs correlate with poor health outcomes such as aging, asthma, amyotrophic lateral sclerosis, frontotemporal dementia, muscular dystrophy, and cancer. Thus, understanding the mechanisms regulating SGs and their functional roles in the cell is paramount to treating disease and promoting extended health spans. Though SGs are known to involve extensive networks of protein-protein and protein-RNA interactions, it remains poorly understood how intermolecular RNA-RNA interactions contribute to SG organization and function. Therefore, the proposed research objectives focus on determining how the SG scaffolding protein G3BP1 facilitates the formation of intermolecular RNA-RNA interactions, what RNA elements compose these interactions, and how the cell mitigates stable RNA-RNA interactions during normal cell function of stress recovery to prevent progression into disease states. In this work, approaches using biochemical, transcriptomic, high-throughput genetic screens, and cancer cell progression models will 1) determine the biochemical features required for RNP granule scaffolding proteins to form intermolecular RNA-RNA interactions and their impacts on stress response, 2) characterize the RNA elements involved in forming stable RNP granule RNA networks, and 3) identify the cohort of proteins responsible for limiting intermolecular RNA-RNA interactions and their roles in preventing progression from healthy to disease states. In the K99 phase, mentorship from Dr. Roy Parker and Dr. Robin Dowell will provide valuable training in biochemical and high-throughput sequencing techniques required to determine the mechanisms and features driving RNA network formation within stress granules. Further support from Dr. Jennifer DeLuca will provide skills in working with cancer progression models to determine the functional effects of SG RNA network dysregulation. The training in biochemical, sequencing, computational, and cell-biological techniques during the K99 phase will be essential to the proposed research and develop valuable skills required for the independent R00 phase. Ultimately, the results from this proposal will advance our understanding of SGs and RNP granules generally by revealing the fundamental role of RNA-RNA interactions in granule organization and maintenance.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Advances in molecular simulation models and methods have made it possible in many cases to simulate biological macromolecules and their environments over time scales of physical relevance with near-quantitative accuracy. In particular, such approaches are now used directly in the pharmaceutical industry to guide synthesis of new compounds using computed changes in small molecule binding affinities. However, despite substantial work by many researchers over decades, the majority of protein-ligand binding phe- nomena related to human health remain prohibitively expensive to model with sufficient accuracy using current simulation capabilities. Such therapeutic modalities include targeting protein-protein interfaces, kinases and phos- phatases inhibitors, and partners of short peptide interaction motifs. One significant reason for this difficulty is that these targets favor bigger and more flexible binders, with both larger scale motions and less frequent transi- tions between metastable states, requiring significantly more sampling than is currently computationally feasible. Additionally, existing force fields have been developed in a piecemeal fashion, do not generalize over all of rel- evant chemistry, and the proper trade-offs between increased complexity and computational cost of force field improvements are not clear. We propose to address these challenges building on our extensive expertise in methods development and simu- lation software for improved biophysical modeling and drug design. In collaboration with other researchers in the Open Force Field Initiative, my group will work to build systematically improvable force fields that achieve high accuracy and broad coverage of NIH-relevant chemistry, that self-consistently model heterogeneous biomolecular systems, and that can be broadly applied across a range of high-performance software packages. In particular, we will use new sources of thermophysical data to optimize and validate behavior in complex chemical environments and will develop statistical approaches to better gauge the utility of increasing force field complexity. In addition, we will develop novel, well-validated molecular simulation sampling methodologies that can capture the full configurational ensembles needed to accurately compute binding free energies of large, flexible ligands. Such algorithms will be designed to support new and anticipated computing architectures such as heterogeneous clusters and cloud computing via asynchronous approaches, and will augment molecular dynamics with recent developments in machine learning structural prediction. This research is significant and transformative as the computational concepts and technology developed via this research will enable accurate prediction of larger, flexible ligands from across all of bioorganic chemistry. The effort has the potential to assist drug developers break away from existing design paradigms of relatively small, rigid molecules bound to relatively rigid well-defined binding sites, accessing new targets and mechanisms of action and as well as therapeutics targeting them.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY The commensal bacteria that inhabit our microbiomes have a direct impact on human health. Recent studies have shown that bacteria in the gut can even alter patient responsiveness to immune checkpoint inhibitors, the latest generation of anticancer immunotherapy. The mechanisms of how bacteria influence the immune response to cancer are poorly understood; therefore, understanding bacterium-human interactions at the molecular level is critical to our understanding of the immune system, disease, and therapeutics. An important way that bacteria influence human cell signaling is through the production of small molecules that activate or inhibit human cellular receptors. We are specifically focused on how diverse bacteria manipulate the mammalian cGAS-STING pathway, a clinically relevant pathway that is critical for the immune response to cancer, as well as viruses and pathogenic bacteria. In humans, cGAS generates a cyclic dinucleotide second messenger that activates STING, which is the crucial step for initiating anticancer signaling. We recently made the unexpected finding that bacteria of the gut microbiome encode enzymes homologous to cGAS. Bacterial cGAS synthesizes cyclic dinucleotides that agonize STING, and cyclic dinucleotides released by bacteria can be taken up by mammalian cells. Cyclic dinucleotides thus provide a molecular basis for bacteria to manipulate the human cGAS-STING pathway and offer a shared molecular language for crosstalk between the microbiota and mammalian cells. Although bacterial and mammalian cGAS are highly similar, there are important differences in the heterogeneity of their cyclic dinucleotide products and studying these differences will decipher the crosstalk between these domains of life. Mammals synthesize one potent second messenger while bacterial cGAS-like enzymes are highly diverse. Different bacteria produce unique cyclic dinucleotide products that vary in their ability to agonize STING and additional human immune pathways. This proposal investigates the hypothesis that bacteria in the microbiome expressing different bacterial cGAS alleles can alter host STING signaling, the host immune response to cancer, and the effectiveness of cancer immunotherapy. First, we will investigate the role of specific bacteria expressing cGAS homologues in altering effectiveness of cancer immunotherapy. Next, we will interrogate known bacterial strains that alter patient responsiveness to cancer immunotherapy for production of cyclic dinucleotides. Finally, we will explore the human immune pathways, including STING, that are activated by the full range of diverse cyclic dinucleotides synthesized by bacteria. This proposal is focused on the mechanisms by which bacteria alter patient responsiveness to cancer immunotherapy; however, I expect results from these studies to be broadly relevant to our understanding how specific bacteria in the human microbiota influence human health and will advance the clinical utility of cyclic dinucleotides as therapeutic agents.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY This advanced technology grant focuses on enhancing cryogenic sample time-resolved electron microscopy (cryoTREM) through the development of a patterned LED/LCD-based illumination system capable of capturing multiple temporal states on a single EM grid with 4 orders of magnitude time range from 1 to 10,000 milliseconds. This system will be developed and deployed in producing time-resolved data sets for several different biological systems with intermediate states with varying degree of conformational magnitude. The resultant data will be integrated with a variety of data processing methods to determine ensembles of time-resolved structures, which can be leveraged to understand molecular mechanisms. These time-resolved EM experiments will be conducted on multiple biological systems and processes including ligand-gated ion channels, a lipid transferase, the V- ATPase, and synaptic vesicle fusion.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Drug overdose deaths have surged more than fivefold over the last two decades, and more than 75% involve opioids. Both rewarding aspects of drug use as well as the avoidance of aversive withdrawal symptoms contribute to opioid addiction. A key question amidst the opioid epidemic is whether rewarding and aversive aspects of opioid addiction can be leveraged to aid recovery in addicted individuals. Opioid drugs of abuse exert their powerful addictive properties by exploiting the brain’s reward center, the ventral tegmental area (VTA). Canonically, the rewarding effects of opioids arise through activation of μ-opioid receptors on VTA neurons that release the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Opioid binding on these GABAergic neurons leads to increased dopamine release in the nucleus accumbens that is critical for opioid reward and opioid-seeking. Surprisingly, we recently discovered μ-opioid receptor VTA neurons that release the excitatory neurotransmitter glutamate. Contrary to the classically rewarding circuit governed by GABAergic neurons, opioid binding on glutamatergic VTA neurons reduces excitatory drive onto dopamine neurons. My preliminary data indicate that GABA release from μ-opioid receptor VTA neurons supports opioid-induced reward, but glutamate release from μ-opioid receptor VTA neurons does not. Based on these findings, we ask two questions: (1) Do glutamatergic or GABAergic μ-opioid receptor VTA neurons alone modulate nucleus accumbens dopamine? and (2) Do glutamatergic or GABAergic μ-opioid receptor VTA neurons alone drive opioid-seeking? We hypothesize that glutamatergic and GABAergic μ-opioid receptor VTA neurons differ in their modulatory effects on the nucleus accumbens reward system and in their ability to drive opioid-seeking behavior. In Aim 1, I propose to specifically activate μ-opioid receptors on glutamatergic or GABAergic VTA neurons while measuring dopamine release in the nucleus accumbens in mice. In Aim 2, I will determine whether selective μ-opioid receptor activation on glutamatergic or GABAergic VTA neurons drives opioid- seeking in a model of relapse. The completion of these Aims will use cutting-edge techniques such as cell type-specific pharmacology (DART), fiber photometry of dopamine sensors (GRABDA), and a reinstatement/relapse paradigm of intravenous self-administration. The mentoring committee at the University of Colorado Boulder, Colorado State University, and Duke University will provide expert guidance throughout the study. I will receive significant training in intracranial microinjections and the methodology of DART, intravenous self-administration and reinstatement paradigms, and the interpretation of GRABDA dynamics. The findings of this study may redefine the neurobiological underpinnings of opioid-induced reward and relapse, which will prove crucial in the continued struggle with the opioid epidemic.