University Of Colorado Denver

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY Prenatal nicotine exposure (PNE) remains a pressing public health concern. At least 10 percent of pregnant individuals in the US report smoking cigarettes (or e-Cigs) during gestation. PNE is linked to neurodevelopmental disorders such as attention-deficit hyperactivity disorder and autism spectrum disorders. Nicotine, via cholinergic nicotinic acetylcholine receptors (nAChRs), enhances cholinergic activity in the prefrontal cortex (PFC), dysregulating development and increasing the risk for PFC-linked neurodevelopmental disorders. PFC-linked neurodevelopmental disorders point at disruptions in synapse maturation leading to synaptic dysfunction. However, the mechanisms by which nAChR signaling, and nicotine exposure, influence synapse maturation are unknown. Our preliminary studies found that cholinergic denervation in the developing PFC impairs excitatory synapse maturation. Additionally, we found nicotine treatment promoted synapse maturation in the absence of glutamatergic activity. This proposal is composed of two aims which seek to test the hypothesis that postsynaptic nAChR signaling modulates excitatory synapse maturation in the developing PFC. The first aim uses a combination of innovative techniques including two-photon uncaging of glutamate, chemogenetics, electrophysiology, and pharmacology to determine if the up-regulation of endogenous cholinergic activity promotes synaptic maturation. The second aim uses two-photon, two-color uncaging of nicotine and glutamate, electrophysiology, and pharmacology to elucidate the synaptic and molecular mechanisms involved in the nicotinic signaling-mediated maturation of synapses. In sum, the studies outlined in this proposal will shed light to the cellular, synaptic, and molecular mechanisms involved in the nicotine-induced maturation of excitatory synapses. Results from this project will advance efforts towards mitigating the impact of neurodevelopmental disorders that result from PNE.

NIH Research Projects · FY 2025 · 2024-02

Abstract Type 1 diabetes (T1D) is caused by an autoimmune response that targets and destroys insulin-producing beta cells in the pancreas. Despite advancements in managing T1D, identifying specific antigens critical for T1D onset has been challenging. Recent research has uncovered hybrid insulin peptides (HIPs), which induce an autoimmune CD4 T cell response and may serve as specific autoantigens in T1D. HIPs are generated when a peptide fragment of proinsulin binds to a peptide fragment from other beta cell proteins. Presence of various HIPs has been validated through mass spectrometric analyses of human and murine islet samples. Several of those identified HIPs have a known CD4 T cell that specifically targets them in human and murine disease. We consider these HIPs as disease-relevant because we not only have mass spectrometric evidence for their existence, but also immunological evidence verifying their role in disease. In this proposal, we plan to study the role of disease-relevant HIPs formed by the enzyme Cathepsin D (CatD) in the progression of diabetes in non- obese diabetic (NOD) mice, a amahor animal model used for the study of T1D. We hypothesize that a mutation of an insulin leucine residue (targeted by CatD) can effectively prevent CatD-mediated HIP formation, resulting in a reduction of disease incidence in genetically modified mice compared to wildtype mice. We will assess HIP formation by analyzing mouse islet samples using mass spectrometry, confirm HIP content through T cell assays with HIP-reactive T cell clones, and monitor mice for disease incidence and immune cell infiltration of the pancreatic islets. The researchers will also investigate the origin of another subgroup of HIPs (identified in human islets) that do not form through a CatD-mediated process. For this we apply a proteomic strategy to probe human islet samples by mass spectrometry with the objective to identify the enzyme responsible for their formation. The results of these experiments will provide a better understanding of the role of HIPs in disease onset. Furthermore, the identification of additional HIP-forming processes in human islets may lead to the discovery of a new therapeutic target, that may allow us to selectively block formation of HIPs in beta-cells. The proposed studies also offer me the opportunity to expand my understanding of several fields including immunology, proteomics, and mass spectrometry. These combined skillsets will provide me with expertise needed to study numerous diseases and pathogenic process throughout my anticipated career as independent investigator.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Natural killer (NK) cells are effector lymphocytes in the innate immune system. They destroy transformed and pathogen-infected cells mainly through secreting the contents of membrane-enclosed cytolytic granules, secretory lysosomes containing the pore-forming protein perforin and the serine proteases granzymes. When a NK cell recognizes its target cell, the contact area between the NK cell and the target cell forms a highly organized structure known as the immunological synapse. Cytolytic granules then migrate toward the immunological synapse, where they fuse with the plasma membrane to release their cytolytic molecules, a process known as cytolytic granule exocytosis. Once released from the NK cell, cytolytic molecules enter the target cell and trigger cell death. Imbalances in cytolytic molecule exocytosis cause major forms of human disorder such as immunodeficiency and chronic pathogen infection. The molecular basis of cytolytic granule exocytosis in NK cells is still poorly understood. The proposed research aims to bridge this knowledge gap. In our preliminary studies, we established an assay to quantify cytolytic granule exocytosis in a physiologically relevant NK-like cell line. Furthermore, we developed new platforms to genetically dissect complex exocytic pathways in mammalian cells using unbiased CRISPR screens. In this research, we will take strategic advantage of these assays to dissect cytolytic molecule exocytosis in NK cells using a genome-wide CRISPR screen. Next, we will validate the candidate genes identified in the screen using a pooled secondary screen. Finally, we will validate selected candidate genes using individual gene knockout. If successfully accomplished, this proposed research will offer the first genome-scale view of the cytolytic exocytic pathway and will significantly broaden our knowledge of NK cell functions. Insights acquired from this work will facilitate the development of novel therapeutic strategies for immune diseases caused by defective cytolytic granule exocytosis.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Despite the prevalence of advanced age as a risk factor for many diseases, its features are rarely replicated in experimental modeling of these diseases. Mimicking elements of aging is particularly rare in the in vitro context, even though in vitro approaches can offer a degree of tunability and control that enable pursuit of unique mechanistic, biological questions. Many diseases also present with sexual dimorphism, wherein men exhibit different disease characteristics than women. Again, this element of sex-specific behavior is rarely examined in the in vitro environment. In aortic valve stenosis (AVS), advanced age and male sex are the top two risk factors; AVS also exhibits sexual dimorphism in its presentation. The only approved treatment for this prevalent disease is surgical valve replacement, a shortfall that is, in large part, due to our relatively poor understanding of its pathogenesis and progression. Current platforms are not set up to query why aging is the strongest risk factor for this disease, or why men exhibit difference pathology than women. Thus, we propose to create in vitro tissue-engineered models that incorporate both age-related and sex-specific features in order to elucidate mechanisms responsible for the onset of AVS hallmarks. Specifically, we will: 1) Characterize age-related changes in valve structure and hemodynamics and create corresponding engineered environments, 2) Evaluate the roles of cellular aging vs. environment aging in sensitizing the valve to pathological stimuli, and 3) Determine how age-related sex hormone changes (and cellular memory thereof) influence the valvular response to pathological stimuli. This study will yield multiple advancements, particularly with respect to in vitro modeling of aging and pathologies, and identification of the most influential age-related features that guide the responsiveness of valve tissues to pathological stimuli. Such work has the potential to identify specific age-related changes and signaling pathways that may be targets for intervention strategies to reduce AVS risk.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT Many fundamental aspects of our lives heavily rely on an intact motor system: we need it to explore, to communicate, to interact with our surroundings. The cerebellum is uniquely involved in reach dysmetria, whereby the limb oscillates about an end target, resulting in diminished reach precision. Though our ability to make precise, goal-directed movements is so critical, we still lack a complete understanding of how the brain generates the appropriate activity to achieve something as simple as reaching for a coffee cup. Fortunately, the cerebellar circuitry is uniquely organized and well-defined; this lends itself to being a tractable structure for testing theoretical models of its computational logic in practice with the advent of updated neuroscientific tools. Two such models that describe how the cerebellar cortex guides endpoint precision in reaching are that it 1) integrates limb velocity to estimate positional values or that it 2) recodes sensorimotor information into temporally distinct activity patterns. To investigate the computational logic the cerebellum engages to enable goal-directed reaching, I will use two-photon calcium imaging to measure activity in early cerebellar cortex during a multi-target reach task in mice. In Aim 1, I will image granule cell axons and molecular layer interneurons to test the hypothesis that information about target location and movement goal is relayed to the cerebellar cortex by its mossy fiber inputs. I will leverage multi-view, high-resolution video, supervised body part tracking, and multi- photon imaging to identify how location and limb velocity information content is encoded by granule cells. In Aim 2, I will use targeted photoexcitation of mossy fiber inputs to induce motor adaptation to test the hypothesis that stimulation results in a misrepresentation of sensory estimates in granule cells, which drives errant reaching behavior, and adaptation to the perturbation is a resultant of a re-mapping of the new information content. Collectively, these data will distinguish whether the cerebellar cortex computes a control function or temporal basis set model to guide endpoint precision. By rigorously evaluating behavioral motor motifs in rodent models and linking these to neuronal activity, we can gain clarity into how discrete actions lead to overall performance and open up more possibilities to better addressing fundamental questions relating to the neural mechanisms of motor control and learning.

NIH Research Projects · FY 2026 · 2024-01

Summary Numerous brain disorders and diseases are caused by deficits in synapse function. Synaptic transmission is traditionally thought to be governed by the amount of neurotransmitter released and the number of neurotransmitter receptors localized to the postsynaptic membrane. However, accumulating evidence suggests synaptic function not only depends on the number of postsynaptic receptors, but also their precise nanoscale positioning within the postsynaptic membrane. For example, at excitatory synapses AMPA-type glutamate receptors (AMPARs) form one or more clustered sub-structures within the postsynaptic density that are precisely aligned with presynaptic neurotransmitter release sites. Modeling studies predict this pre/post alignment to be critical for efficient AMPAR activation, but this has been challenging to address experimentally. The degree to which receptor nano-positioning influences synaptic function remains unclear due to a lack of suitable approaches for 1. Rapid and reversible perturbations to synaptic nanostructure that allow simultaneous synaptic function measurements. 2. Probing glutamate concentrations directly within distinct synaptic nanodomains. 3. Testing the functional relationship between receptor activation and distance from neurotransmitter release sites. To address these issues, we have recently developed new approaches for rapidly manipulating postsynaptic scaffolds and receptors in real time. In parallel we have developed a new set of genetically encoded affinity reagents for labeling and manipulating endogenous AMPARs. We propose to combine these approaches to assess the functional relevance and regulation of nano-scale positioning within the PSD, a problem that has been challenging to assess using conventional approaches.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Nervous system development and regeneration relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells. Though neuronal and glial differentiation is extensively studied, less is known about the molecular signals that modulate the transition from neurogenesis to gliogenesis. Oligodendrocytes, glial cells that support axon conduction and survival through myelination, are essential for nervous system development and regeneration. During spinal cord development, distinct neural progenitors in the pMN domain undergo a neuron-glia switch to sequentially produce motor neurons followed by oligodendrocyte progenitor cells (OPCs). Similarly, successful regeneration after spinal cord injury requires the coordinated production of both neurons and oligodendrocytes to functionally restore damaged circuits. Zebrafish are highly regenerative compared to mammals and endogenous neural progenitors produce motor neurons after injury, but interestingly do not produce OPCs. The molecular mechanisms that regulate the neuron-glia switch to specify neural progenitor cells into a neuronal or oligodendrocyte fate during development and after injury remain unknown. Recently, our lab conducted fate mapping and single-cell RNAseq and revealed a molecularly distinct subset of pMN neural progenitors, pre-OPCs, that are specified to commit to the oligodendrocyte lineage. Our data revealed that pre-OPCs are uniquely enriched for factors involved in Wnt signaling, a cascade that modulates OPC differentiation during development and that is reactivated after injury to regulate neurogenesis. Using zebrafish, I will utilize immunohistochemistry and transgenic imaging techniques alongside CRISPR/Cas9 mutagenesis strategies to determine how axin1 modulates wnt expression in pMN progenitors to activate a transcriptional switch in tcf7l2 and drive a neuron-glia switch to specify pMN progenitor cells into pre-OPCs. Further, through powerful single-cell multi-omics techniques I will identify transcriptional interactions with neuronal and oligodendrocyte-specific genes in pMN progenitors to establish a gene regulatory network that drives the neuron-glia switch. Finally, I will use pharmacological and genetic tools to manipulate wnt expression after spinal cord injury and induce a neuron-glia switch in vivo to drive gliogenesis in endogenous neural progenitor cells. Together, these studies will uncover molecular regulators of pMN progenitor specification and highlight the role of the neuron-glia switch in nervous system development and regeneration.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin-producing β-cells in the pancreatic islets. Several theories have been proposed to explain T1D pathogenesis including aberrant immune cell activity as well as increased β-cell sensitivity to inflammatory and metabolic stressors. It has become increasingly apparent that β-cells may not be passive immune cell targets but can contribute to the disease intrinsically. However, it remains unclear whether the risk of developing T1D is already introduced during β-cell formation. Postnatal β-cell maturation may be a T1D relevant β-cell developmental process, not only because it occurs during the peak incidence of T1D diagnosis but also because it determines whether an individual has an adequate functional β-cell pool to cope with environmental stressors. However, the mechanisms underlying human β-cell maturation have been understudied due to the absence of an appropriate human β-cell model. While human pluripotent stem cell (hPSC)-derived β-cells (SC-β-cells) hold immense potential for modeling T1D, current production methods do not permit proper functional maturation, hampering their utility in studying β-cell maturation. One of the hallmark features of mature β-cells is glucose-induced mitochondrial respiration, which is lacking in SC-β-cells. It has been suggested that environmental cues, such as nutrients and circadian rhythm, drive β-cell maturation and establish a mature β-cell state through gene expression regulation and epigenetic modifications. Our objective is to uncover gene regulatory mechanisms that underlie the acquisition of glucose- dependent mitochondrial function in human β-cells and assess the suitability of SC-β-cells as a model for studying β-cell maturation. This work will leverage the genomic and genetic methodologies that we have previously developed for SC-β-cells, including a platform for genome-wide functional perturbation screening, single-cell genomics assays, and a computational tool to infer gene regulatory networks. Expanding on our preliminary studies, we propose to investigate and modify key transcriptional programs governing SC-β-cell mitochondrial activity and induce maturation in vitro (Aim 1). Mechanistically, we plan to investigate the correlation between changes in SC-β-cell function and the epigenome that occurs during the maturation process (Aim 2). It has been established that SC-β-cells exhibit improved function following engraftment into mice. Thus, in the second aim of our study, we will also investigate epigenetic alterations that occur during this in vivo maturation process. We anticipate that our in vitro manipulation of key transcriptional programs will induce similar epigenetic remodeling as observed in vivo. Together, our proposed study will advance the culture of SC-β-cells and demonstrate their utility as a model for investigating human β-cell maturation.

NIH Research Projects · FY 2026 · 2024-01

Abstract Background: Following surgical resection for intractable epilepsy caused by malformations of cortical development (MCDs), over 30% of children continue to have life-changing seizures. Mutations in genes of the mechanistic target of rapamycin (mTOR) pathway lead to these disorders that include focal cortical dysplasia (FCD), tuberous sclerosis complex and hemimegalencephaly. Therefore, there is a critical need to discover the specific mechanisms by which increased mTOR signaling leads to the development of epilepsy to develop specific therapies for children with MCDs. The central hypothesis of this proposal is that mTOR pathway upregulation in pyramidal cells causes axon overgrowth, creating an epileptogenic network in both mouse and human MCDs Methods: The first step in testing the central hypothesis will be to determine the effect of mTOR pathway upregulation on axonal length in mouse FCD and human MCDs via quantifiable analysis of fluorescently labeled murine axons of and by 3D reconstruction of individually filled human neurons. A second set of experiments will be performed to determine whether some pyramidal neurons act as operational hub cells in human and mouse MCDs. Calcium imaging on ex vivo brain slices of mouse and human tissue will be used to define the local network connectivity and identify hub cells. Finally, axonal degeneration will be induced in vivo in dysplastic cells in mouse FCD to determine the effect of targeted reduction in axon length on seizure frequency. Implications: The proposed research will help to determine whether axonal overgrowth is a key mechanism by which mTOR upregulation leads to the generation of pharmacoresistant epilepsy in children with MCDs. Additionally, this project will demonstrate whether targeting axonal overgrowth offers the potential for treatment of seizures. Thus, the results of this research will provide further biological understanding of MCD and provide hope for rational development of future successful therapies for children with this set of disorders. Career Development Plan: During the five years of this award, Dr. Alexander will work with a team of mentors to achieve six specific career goals which allow her to accomplish this project and will also cement her transition to independent investigator. With hands-on mentorship and a focused set of coursework, she will work towards three training goals. These are as follows: 1) to obtain training and mentorship in the principles and techniques of molecular neuroscience; 2) to obtain mentorship and training in neuronal network analysis using calcium imaging; and 3) to obtain training and mentorship in laboratory management as a neurosurgeon- scientist. Dr. Alexander is a pediatric neurosurgeon at Children's Hospital Colorado. Her chairman and division chief continue to support 50% protected time through on-campus resources which include dedicated research space, a rodent EEG core, shared equipment and a startup package for laboratory equipment and personnel.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY/ABSTRACT The prevalence of adolescent obesity continues to rise, with parallel increases in obesity-related comorbidities such as cardiovascular disease (CVD) and type 2 diabetes (T2D). Our lab has demonstrated that youth onset T2D (Y-T2D) appears to be a more aggressive disease than in adults, thus dedicated studies investigating comorbidities of obesity such as CVD and T2D are critical. Epicardial adipose tissue (EAT) is a visceral fat depot that has anatomical proximity to the coronary arteries surrounding the heart and has been suggested as an independent cardiometabolic risk factor in adults. EAT is a highly active metabolic tissue that secretes pro- inflammatory factors and therefore is reasonable to expect EAT to have deleterious consequences in cardiac health. However, the relationship between EAT and CVD in adolescents is less studied. Further, measurement of EAT has been commonly performed using echocardiogram, which can result in poor image quality when compared to gold standard MRI, or computed tomography which includes radiation risk. Thus, it is critical to address early indicators of CVD such as EAT in adolescents with obesity and T2D. We propose to use novel cardiac MRI methods to assess cardiac structure and function, including EAT. Further, it is important to study the impact of diabetes treatments on cardiometabolic risk factors. Specifically, metabolic and bariatric surgery (MBS) is now under investigation in youth with severe comorbidities of obesity and appears to elicit significant weight loss, however there is less data on its impact on overall cardiovascular health in adolescents. MBS is also being studied for its weight loss independent effect on metabolism and health, including on the incretin hormone glucagon-like peptide-1 (GLP-1) which has recently been shown to improve glycemic control and even facilitate weight loss. Therefore, the goal of this proposal is to investigate the potential independent relationship between EAT and cardiometabolic health in Y-T2D and explore changes in EAT as a potential mediator of changes in cardiometabolic health in response to MBS. To address this goal, the first aim of this proposal will be to determine if baseline MRI-assessed EAT in Y-T2D correlates with cardiometabolic health, independent of BMI. Adolescents undergoing MRI of the heart and aorta will have EAT volume measured and related to cardiometabolic health. The second aim of this proposal is to determine the impact of MBS on EAT volume in adolescents with T2D and if improvements are independent of weight loss, with the potential improvements mediated through GLP-1 secretion. To summarize, this dedicated study in adolescents with severe obesity and Y-T2D will add to the current gap in knowledge as there are no publications to-date examining the effect of MBS on EAT, nor on cardiac or vascular structure or function, nor on the relationship between EAT and cardiometabolic health in Y-T2D.

NIH Research Projects · FY 2026 · 2023-12

PROJECT ABSTRACT Kidney transplantation is the desired treatment for kidney failure. While short term kidney transplantation outcomes have improved significantly, long-term outcomes remain suboptimal. Death with functioning graft from cardiovascular disease (CVD) is a major cause of graft loss. The annual rate of cardiovascular events in kidney transplant recipients (KTRs) is 50 times higher than that of the general population. Over 30% of KTRs have pre-existing type 2 diabetes (T2D) and around 10-40% will develop posttransplant T2D. T2D magnifies risk for CVD, graft loss and mortality among KTRs. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are now standard of care in patients with chronic kidney disease (CKD). Several large randomized controlled trials (RCTs) have established that SGLT2i improve kidney and cardiovascular outcomes. Using single-cell RNA sequencing (scRNA-seq) data from kidney biopsies we found that SGLT2i treatment benefits the diabetic kidney by mitigating diabetes-induced metabolic perturbations via suppression of mammalian target of rapamycin complex1 (mTORC1) in kidney tubules. Additional preliminary data from our group suggest that SGLT2i is associated with improved kidney oxygenation by kidney MRI, which may contribute to the beneficial kidney effects of SGLT2i. These potential mechanisms are particularly promising in KTRs, as our preliminary studies in KTR biopsies found enrichment for renal metabolism and mTORC1 signaling post-transplantation. mTORC1 plays a key role in the immune response and graft rejection in KTRs. SGLT2i may have benefits for KTRs, such as improving graft function and lowering the risk of CVD. However, the potential benefit of SGLT2is for KTRs has not been explored in RCTs. Accordingly, we propose a 12-month, randomized, placebo-controlled, double-blind study in 80 adult KTRs with pre-existing T2D or new onset posttransplant diabetes with eGFR 30-90 ml/min/1.73m2 and urine albumin-to-creatinine ratio (UACR) of 30-5000 mg/g to examine the effects and underlying mechanisms of dapagliflozin on kidney and cardiovascular structure and function. The primary outcomes are change in UACR and establishing safety. In Aim 1, we will assess the effects and safety of 12 months of dapagliflozin vs. placebo on kidney graft structure and function via kidney MRI as well as kidney allograft biopsies in a subset of participants (n=40). In Aim 2, we will determine the effects of 12 months of dapagliflozin vs. placebo on arterial stiffness, cardiac structure and function assessed by aortic pulse wave velocity and aortic and cardiac MRI. In Aim 3, we will implement state-of-the-art interrogation of kidney tissue from baseline and 1-year biopsies by integrating scRNA-seq with detailed morphometry as well as comprehensive clinical phenotyping to investigate the mechanisms of dapagliflozin in KTRs, and associations of these mechanisms with kidney structure and function. The expected results will provide critical insight into the efficacy, safety and mechanisms of dapagliflozin in promoting kidney graft function and cardiovascular health in KTRs.

NIH Research Projects · FY 2026 · 2023-12

ABSTRACT This submission by Hillary Lum, MD, PhD for the NIH Midcareer Investigator Award in Patient-Oriented Research (K24) enables Dr. Lum to: 1) mentor junior clinical investigators in dementia care research, and 2) enhance the science of community-engaged research and co-design methods involving persons affected by Alzheimer's Disease and Related Dementias (ADRD). Dr. Lum is a geriatrician, palliative medicine physician, and prior Beeson Scholar with an independent ADRD research program, ADRD team-science collaborations, a national reputation in ADRD palliative care research, and a strong record of grants and publications. Mentorship: Over the last decade, Dr. Lum has mentored more than 30 residents, fellows, and junior clinical investigators in geriatrics, palliative care, neurology, and other patient-oriented aging disciplines. She has research mentorship leadership roles including serving as Co-Director of the NIA T32 Multidisciplinary Palliative Care in Aging Post-Doctoral Fellowship Program and Associate Director of the NCATS Clinical Faculty Scholars Program at the University of Colorado. Her mentoring objectives are to increase the pipeline of ADRD and geriatric investigators who progress to independence, and to lead institutional and national ADRD and geriatric patient-oriented research mentorship programs. Research theme: Dr. Lum's research integrates community-engaged research and co-design methods into real-world intervention design, testing, and implementation to improve care for persons living with dementia and family care partners. Community-engaged research is the process of working collaboratively with patients and others during conceptualization, conduct, and dissemination of research. Co-design methods, focusing on developing interventions, are part of community engaged research. As an example of community-engaged research in ADRD, Dr. Lum convened a “Memory Tech Council” of patients, care partners, and community members to identify priorities for use of consumer health information technology embedded in the health system to improve dementia care. This K24 proposal will provide Dr. Lum with protected time to advance the science of engagement in ADRD by focusing on: 1) expanding capacity for junior investigators to integrate engagement methods into their ADRD research; 2) identifying community-engaged research methods for partnering with persons living with dementia and care partners; and 3) organizing co-design tools for dementia care interventions into a new online resource for dementia care researchers. Impact: Through this K24 award, Dr. Lum will enhance the science of engagement in ADRD, enhance her mentorship through current and new research mentorship roles at University of Colorado and nationally, and enhance the field through a pipeline of clinical investigators in ADRD and patient-oriented aging research.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY The composition of microorganisms that inhabit the gut, known as the gut microbiome, has been strongly associated with the pathogenesis of several diseases including cancer, diabetes, and multiple sclerosis. Notably, several high-profile studies have reported that the composition of the gut microbiome is correlated with responses to immune checkpoint inhibitors (ICIs) in human cancer patients. While these studies have proposed several mechanisms accounting for this phenomenon, a specific set of cellular and molecular interactions that explains these responses remains to be elucidated. One possible mechanism is through the immunologic recognition of the byproducts of microbial metabolism. Mucosal-associated invariant T (MAIT) cells are an abundant, highly conserved subset of innate-like T cells that recognize products of microbial riboflavin synthesis presented by the non-polymorphic HLA-like molecule, MR1. Mice grown in germfree conditions have a significant reduction in MAIT cells and the introduction of bacteria with high production of riboflavin drastically increases MAIT cell frequency and activation. Our preliminary data indicate that MAIT cells are found in human melanoma tumors. Furthermore, we found that patients with stage III/IV melanoma have significantly decreased frequencies of MAIT cells amongst their PBMCs, which can be reversed upon anti-PD-1 immunotherapy treatment in responsive patients but not in non-responsive patients. Most strikingly, we observed that patients with high frequencies of circulating MAIT cells had significantly improved overall survival compared to those with low frequencies. Furthermore, we have found that the tumors of responding patients have increased expression of MR1 mRNA compared to non-responders. Interestingly, responsive patients also show increased relative abundance of riboflavin-synthesizing bacteria in their gut microbiome compared to non-responders. Additionally, in our novel MAIT cell deficient mouse model, syngeneic melanoma tumors grow significantly faster and antitumor immunity is significantly diminished compared to wild type mice. Key questions arise from these observations. First, how are riboflavin biosynthesis and MAIT cell functions influenced by ICI treatment? Additionally, what role do MAIT cells play in tumor immunity and what therapeutic benefit can MAIT cell-directed therapies offer when combined with ICI treatment? We hypothesize that MAIT cells are required for anti- melanoma immunity and that activated MAIT cells link the microbiome and tumor immunity through microbial riboflavin synthesis. This hypothesis will be addressed in the following Specific Aims: (1) Determine the mechanism underlying the association between MAIT cells and the dynamics of riboflavin-synthesizing microbes in the gut microbiome during immune checkpoint inhibitor therapy; and (2) Define the contributions of MAIT cells to ICI therapy responses. Should this study reveal a role for MAIT cells in anti-tumor immunity that is dependent the microbiome, modalities aimed at expanding and/or activating MAIT cells during therapy may provide substantial clinical benefit for melanoma patients.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Infection with a pathogen can have a significant impact on drug metabolism and disposition in the host and can even determine treatment outcomes. This “pathobiome”, meaning the total amount of pathogenic microbe present during an infection, may have a profound impact on drug metabolism in the disease state when individuals are much more likely to receive drugs, particularly antibiotics. Despite the mounting evidence, we still understand little regarding the capacity of pathogens to contribute to drug metabolism in the host. Understanding the role of pathogens in drug metabolism will fill a gap in our current knowledge and it also has the potential to lead to the identification of novel antimicrobial drug targets. Our preliminary data demonstrates that CYPs from Pseudomonas aeruginosa (PA), an important opportunistic pathogen in the lung of cystic fibrosis (CF) patients, are highly promiscuous and capable of metabolizing drugs - including the antibiotic ciprofloxacin used to treat PA infections. Furthermore, we have also demonstrated that secondary metabolites produced by PA can induce expression of human CYP1A2, potentially leading to increased clearance of ciprofloxacin in the host. This specific application focuses on characterizing the role of CYP enzymes and secondary metabolites from PA. Our central hypothesis is that P. aeruginosa can modulate drug metabolism in the local environment of the lung in order to reduce the effectiveness of drug therapy. In order to test our hypothesis, we propose the following specific aims: 1. Characterize the metabolic capability of P. aeruginosa CYPs to metabolize drugs relevant to the patient CF host; 2. Determine the potency and specificity of P. aeruginosa secondary metabolites to induce expression of host CYP drug metabolizing enzymes in human lung cells, and 3. Identify the structural contributions to substrate and inhibitor specificity for P. aeruginosa CYPs using 19F biomolecular NMR. Characterizing the ability of PA to metabolize drugs in the host will allow us to quantitatively determine the contribution of PA to host drug metabolism at the site of infection and develop inhibitors that may limit PA antibiotic resistance. Understanding the impact of the P. aeruginosa pathobiome contribution to drug metabolism and antibiotic resistance in the host is paradigm shifting in that it will expand our idea of pathogen regulation of host processes and provide new opportunities for potential adjuvant drug targeting in the pathogen to increase the sensitivity to otherwise effective antibiotics.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Cilia damage and ciliated cell loss brought on by epithelial remodeling contributes to defective mucociliary clearance in chronic inflammatory airway diseases such as cystic fibrosis (CF), chronic rhinosinusitis (CRS), asthma and chronic obstructive pulmonary disease (COPD). Proper ciliated cell formation requires coordination of ciliated cell fate acquisition and ciliogenesis programs, but our mechanistic understanding is incomplete. Current treatments do not directly target or fully reverse ciliary dysfunction, highlighting the need for novel molecular targets for specific therapies. We showed that ciliated cell formation and function depend on sequentially deployed Wnt signaling pathways. First, canonical, or β-catenin-dependent Wnt signaling (Wnt/β- cat) is required for ciliated cell fate acquisition, which then must be turned off. Next, noncanonical Wnt/planar cell polarity signaling (Wnt/PCP) is turned on and stays on to control ciliogenesis and polarized ciliary motility. In CF and CRS airways and in primary cultures remodeled by proinflammatory cytokine treatment Wnt/β-cat stays active, and Wnt/PCP is blocked. Wnt/β-cat inhibition alleviates ciliary dysfunction in vitro. Thus, we propose that instead of two independent Wnt pathways, a canonical to noncanonical Wnt signaling switch controls healthy ciliated cell formation, and “switch failure” is a mediator and therapeutic target in ciliary dysfunction. Our preliminary data show that DKK3, a canonical Wnt/β-cat inhibitor and WNT4, a noncanonical ligand, co-secreted by airway epithelial stem cells are required together to suppress Wnt/β-cat and to activate Wnt/PCP within ciliated cells. Downstream, we show that β-catenin, the central effector of Wnt/β-cat is inactivated and sequestered at the basal bodies of cilia. We demonstrate that DKK3 and WNT4 expression is disrupted in diseased, and cytokine treated epithelia. Thus, we hypothesize that DKK3/WNT4-dependent signaling and β-catenin basal body sequestration are required for healthy ciliated cell formation and that disruption of this Wnt switch mechanism mediates in ciliary dysfunction in inflamed airway epithelia. Here, we use transgenic mouse models, human primary cells, and tissues to test this hypothesis in the following aims: In Aim 1, we test the necessity and sufficiency of epithelial DKK3/WNT4 to regulate Wnt signaling and ciliated cell formation through the switch using Dkk3/Wnt4 knockout mice, CRISPR deletion in cells and ectopic expression. In Aim 2, we test if the switch is mediated by the DKK3/WNT4-dependent sequestration β-catenin at basal bodies where it then acts to control ciliogenesis. In Aim 3, we investigate the IL-1β cytokine as a driver of Wnt switch failure and test the ectopic DKK3/WNT4-mediated mitigation of ciliary dysfunction. The new paradigm of the canonical to noncanonical Wnt signaling switch explored in this proposal will establish molecular mechanistic insight into the role of airway epithelial Wnt signaling, elucidate switch failure as a cause of ciliated cell dysfunction, and provide the rationale and potential targets for Wnt modulation in a wide range of chronic lung diseases.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY The hypoxia of high-altitude (HA, >2500 m) increases the frequency of fetal growth restriction (FGR) 3-fold. Reduced uteroplacental perfusion is an important contributor to altitude-associated FGR and is determined, in part, by reduced vasodilatory sensitivity of the myometrial (MyoA) and uterine arteries (UtA). It is therefore vital to define mechanisms that defend uteroplacental blood flow and fetal growth under hypoxic conditions. Using a metabolomic approach we have found maternal and fetal circulating nucleotide metabolites belonging to the purinergic signaling pathway (i.e., adenosine, ADP, AMP, ATP, UDP, UDP-glucose) to differ between HA and low altitude (400 m) in Bolivia, we also observed differences in nucleotide abundance when comparing FGR with uncomplicated pregnancies. Furthermore, our preliminary functional data showed that adenosine vasodilates MyoA and chorionic plate arteries (CPA), the latter are fetoplacental vessels important for the regulation of fetal circulation. In addition, confirming the role of these nucleotides, purinergic receptor agonists known to act as vasconstrictors evoked vasoconstriction in CPA. Thus, we uteroplacental response to nucleotides that elicit vasoconstriction at hypothesize that t he residence blunts vasodilatory responses to vasorelaxant nucleotide metabolites and enhances the vasoconstrictive HA compared to LA. hypoxia of HA Aim 1 will establish the effect of HA pregnancy on the maternal plasma metabolome by mass spectrometry, determine the effect of vasoactive metabolites that differ between altitudes on MyoA vasoreactivity by myography and, using a targeted approach, determine the relationship between the maternal metabolome and UtA blood flow. Aim 2 will determine the effect of HA on the fetal circulation metabolome, the regulation of CPA vasoreactivity by vasoactive nucleotides, and establish the relationship of such effects for fetal oxygenation and growth. In both aims, we will measure protein expression in MyoA and CPA of key purinergic receptors, ectonucleotidases, and enzymes known to participate in purinergic signaling. Our findings will provide the basis for our future research to identify molecular pathways that integrate uteroplacental and fetoplacental perfusion and cellular metabolism during pregnancy to maintain fetal growth.

NIH Research Projects · FY 2026 · 2023-12

This K24 mid-career investigator award in patient-oriented research is to support the mentoring, research, and career development activities of Dr. Tell Bennett. Dr. Bennett is an Associate Professor in the University of Colorado School of Medicine and a practicing pediatric ICU physician and informaticist/data scientist with research concentrations in predictive analytics, electronic health record (EHR) data, and clinical decision support (CDS) tool implementation. He is the Informatics Director for the Colorado Clinical and Translational Sciences Institute (CCTSI) and Vice Chair of Clinical Informatics in the Department of Biomedical Informatics. His combined leadership roles have enabled him to build a rich mentoring environment for patient-oriented informatics research. This K24 application proposes to sustain and grow that mentorship program. He currently mentors clinician-scientists in a variety of clinical fields including intensive care, pharmacy, surgery, endocrinology, malignant hematology, and clinical psychology. The K24 Mentoring Plan aligns with Dr. Bennett’s mission to grow patient-oriented research using informatics and data science methods and tools. The mentoring plan leverages educational, career development, and research support programs available through the CCTSI and the new Department. The K24 Research Plan is to develop and implement machine learning and computational physiology models deployable as EHR-based CDS tools. Dr. Bennett currently leads and mentors projects developing CDS tools in both outpatient and inpatient care settings and in a variety of clinical domains include heart failure, traumatic brain injury, serious bacterial infection and sepsis, COVID- 19, thyroid cancer, and postpartum depression. In this K24, developing CDS tools to improve decision-making and outcomes in children with acute respiratory failure (ARF) is a natural next step in this work. ARF is a common, important, and NIH-relevant condition that causes significant pediatric morbidity and mortality. The aims of the project are to 1) develop and validate a dynamic machine learning-based real-time prediction model for intubation in children with ARF and 2) phenotype current lung injury state in mechanically ventilated children using computational physiology models. These models can be deployed as CDS tools and tested as interventions in future clinical trials to improve patient outcomes. The K24 Career Development Plan includes formal mentorship training and coursework in signal processing and dynamical systems models. This group of skills will make Dr. Bennett a more successful mentor.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT: Dendritic cells (DCs) are sentinels of the immune system that operate at interface of the innate and adaptive immune response. DC-driven responses require their migration from peripheral tissue to the draining lymph node in order prime naïve T cells. Understanding and discovering novel targets that can manipulate DC localization will be beneficial for developing new therapeutic interventions, particularly in the context of vaccination where DC-driven responses are critical for initiation of memory T cell responses. Programmed death ligand-1 (PD-L1) and CD80 are cell-surface protein ligands that belong to the B7 family of proteins and possess the ability to bind to the CD28 family of receptors on T cells in order to provide coinhibitory or costimulatory signals. In addition to their roles in cell-cell communication between DCs and T cells, how PD-L1 and CD80 regulate intracellular signals within migratory DCs remains understudied. Recent published work from the laboratory of my mentor, Dr. Beth Tamburini, demonstrated that PD-L1 intracellular signaling plays an important role in regulating DC migration by controlling chemokine receptor signaling in the context of a type I immune response. My preliminary data suggest that blocking extracellular interactions of PD- L1 with CD80, but not PD-1, decreases DC migration during an inflammatory response within the skin. Additionally, I also found that increased amounts of pathogen-specific tissue resident memory T cells (TRM) persist in the skin after resolution of vaccinia infection in mice with impaired PD-L1 intracellular signaling within cross-presenting DCs. With these preliminary findings in mind, we hypothesize that PD-L1 cis interactions with CD80 regulate intracellular signaling cascades needed to promote migration of DCs. We also hypothesize that while PD-L1-mediated DC retention during cutaneous infection leads to enhanced formation of TRM cells, persistent antigen presentation leads to T cell exhaustion and impaired cell-mediated immunity upon rechallenge. Our aims are as follows: 1. Define which extracellular interactions between PD-L1 and CD80 regulate DC migration and 2. Determine how PDL1 intracellular signals facilitate memory T cell phenotype and function during a viral infection. These studies present an opportunity to investigate the mechanism by which PD-L1 governs migration of cDCs and characterize how retention of migratory DCs in the skin can alter T cell responsiveness to viral antigen. These findings are especially significant in the context of PD-L1 immunotherapy, currently used in the clinic, which prohibits both cis and trans PD-L1 interactions. Loss of these interactions by PD-L1 binding partners could explain resulting off-target effects within nonlymphoid organs such the skin in patients on PD-L1 immunotherapy.

NIH Research Projects · FY 2026 · 2023-12

Effective clinical and translational research (CTR) requires teams of scientists who can move out of traditional siloes to engage in research across the translational ecosystem. Competencies in a discipline known as clinical and translational science (CTS) that includes an array of fundamental characteristics has been proposed by Gilliland and others, which extends beyond traditional CTR characteristics to feature boundary crossing, process innovation, and systems thinking in its repertoire. Notably, CTR training programs have addressed these features in a relatively limited fashion to date that has contributed to the inability of CTR scientists to nimbly adapt to uncertainty and innovation in the research environment. Therefore, for our CTSA K12 Program integrated into Workforce Development of our Colorado Clinical and Translational Sciences Institute, we have incorporated Gilliland’s fundamental CTS characteristics into our K12 program objectives. We request support for six K12 junior faculty scholar positions; appointments will be of at least two years, but no more than three years’ duration. Our K12 program will support development of scholar self- efficacy using guidance from K12 faculty mentor dyads, and support from K12 program directors. Scholars will gain key knowledge, skills, and abilities in an integrated neighborhood of opportunities in CCTSI programs (funded in our UM1 application) and at academic Partnering Institutions. Our K12 program features six objectives to ensure scholars’ academic skills development and enlargement of their information and mentoring network: (1) execution of an independent career development plan based on Gilliland’s fundamental characteristics to provide core CTS knowledge, personalized to the scholar’s research interests and experience, (2) receipt of evidence-informed mentoring training in collaboration with faculty mentors, (3) professional/managerial education, and team leadership training, (4) advanced experiences in academic writing, including dissemination and communication strategies to enhance impact of research, (5) building relationships with key stakeholders and members across the scientific ecosystem, and (6) connection to innovative CTS programs to push existing knowledge boundaries and augment the collaborative network. Planned short- and long-term evaluations and continuous quality improvement of the program will guide efforts and address evolving demands. Importantly, one additional objective for our K12 program will ensure streamlined recruitment processes to augment accessibility of the program to a high number of applicants. Our K12 Program contrasts with other programs at our institution given its heterogeneous, disease agnostic approach, with focus on the training efficient translational scientists who can surmount barriers inevitably present in research itself, and in one’s career. The program will support scholars’ growth to become recognized leaders of diverse research teams with independent extramural funding who can innovate across the translational ecosystem as collaborative clinical translational scientists.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Transgender adults in the U.S. are disproportionately affected by tobacco use. Transgender adults are more than twice as likely to smoke cigarettes than the general U.S. adult population. In addition to the great burden of disease tobacco use places onto this population, tobacco use is also a contraindication for gender-affirming care, a medical necessity for many transgender persons to achieve full mental and physical health. Despite the health-related needs of transgender adults, existing smoking cessation interventions fail to address the specific needs of this population. As such, the National Academy of Medicine and U.S. Surgeon General identifies transgender-specific health needs and developing and testing the effectiveness of interventions for transgender adults a national priority. Despite the role health care providers can play in cessation, transgender adults report barriers to accessing evidence-based, clinician-delivered interventions. A transgender-specific mHealth intervention could serve as a cost-effective and scalable solutions to improving health outcomes for this often hard-to-reach population. Furthermore, such an intervention can be a health services resource for health care providers who might not have the time or expertise to provide gender affirming smoking cessation support to their transgender patients. Building on our team's research expertise in transgender health, mHealth, tobacco cessation, and health systems tobacco-related care, we propose to develop and pilot test an mHealth smoking cessation intervention for transgender adults: Proud to Quit (P2Q). P2Q will provide tailored messages, peer social support, and self-monitoring features. We will develop P2Q by collaborating with an Intervention Advisory Workgroup comprising of transgender adults and providers of gender affirming health care, and implement iterative intervention design processes and usability testing. Through a remote pilot randomized controlled trial, we will assess acceptability and feasibility of an mHealth smoking-cessation intervention for transgender adults as well as its preliminary efficacy using self-report and objective biomarker data. The findings from this proposal will inform the feasibility of and protocols for a full-scale services research effectiveness trial. The project would redress the dearth of smoking-cessation interventions tailored to transgender adults by offering a new tool and the ability to reach transgender adults in diverse locations; it would offer a cost-effective approach that ensures the intervention can be broadly disseminated with fidelity; and, ultimately, it would reduce tobacco-related health disparities among transgender adults, who are disproportionately affected by tobacco use.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Children in families with healthcare communication in a language other than English (LOE) are at increased risk of worse asthma care and outcomes compared to those without a language barrier. There is a gap in knowledge about effectiveness and implementation outcomes of interventions to address asthma disparities for children in LOE families. Asthma navigators play a key role in asthma education and care coordination, resulting in improved asthma outcomes. Our local model of asthma navigation and care coordination to support meeting social determinants of health needs using a lay health worker model has improved asthma outcomes among urban racial/ethnic minority children but has not focused on children in LOE families. To address this gap, we propose, the Navigating Toward Equitable Asthma Management Program (Nav-TEAM) intervention, an adaptation of evidence-based asthma navigation specifically for children in families who communicate in LOE. As with existing asthma navigation programs at our health system, Nav-TEAM will align with EXHALE, the Centers for Disease Control and Prevention National Asthma Control Program compilation of asthma management strategies that have been proven to reduce asthma-related healthcare utilization and costs. The overall scientific goals of this community-engaged study are to: evaluate the effectiveness of Nav-TEAM on pediatric asthma outcomes for 280 children whose families communicate in LOE, evaluate implementation outcomes and application of implementation strategies, and assess cost and contextual factors to support sustained implementation, scale up and scale out. The study will be implemented in a large primary care clinic serving primarily Medicaid-insured children and in subspecialty pediatric pulmonary clinics using the Practical, Robust Implementation and Sustainability Model (PRISM) - inclusive of RE-AIM (Reach, Effectiveness, Adoption, Implementation Maintenance outcomes with an equity lens – as the guiding Dissemination & Implementation Science framework. We will use community-engaged processes to tailor asthma navigation for LOE families in primary and subspecialty pediatric pulmonary care settings at Children’s Hospital of Colorado, a large academic children’s health system serving urban and rural patients. Subsequently, we will conduct a hybrid type 2 effectiveness-implementation trial to simultaneously evaluate the effectiveness of Nav-TEAM at reducing asthma-related emergency department use among children with asthma age 4-14 years using a pragmatic randomized controlled trial as well as a mixed methods assessment of implementation using PRISM/RE-AIM focusing on equity. We will then assess cost, sustainability and contextual domains to understand factors that enable implementation, sustainability, and scale-out of Nav-TEAM. Results from this study will provide information on the effectiveness and implementation of evidence-based asthma navigation adapted specifically for families who communicate in LOE, and will offer lessons that we will capture in an implementation and sustainability guide to promote scaling to other clinical settings.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Long COVID is an important public health threat impacting millions of individuals including 600,000 Coloradans. Current Long COVID care is highly fragmented, variable in nature, and in constant evolution. Creating a novel approach to practice support with continued education and streamlining care for high Long COVID will improve care integration and patient experience. We will implement the Colorado Multidisciplinary Translation Network (CO-MTN) comprised of integrated, multidisciplinary Long COVID care clinics and primary care clinics working together in a tiered care delivery pathway. CO-MTN creates a bidirectional translational care network for integrating expertise, education, referrals, and care between MDCs, PCPs and teams, and their patients. CO-MTN includes 1) the State of Colorado-supported Long COVID Community of Practice comprised of three geographically distributed MDCs, 2) a state-wide practice-based research network representing 280 primary care practices, known as the State Networks of Colorado Ambulatory Practices & Partners, and 3) a proven tele-education system used to provide health care practitioners specialty training, Extension for Community Healthcare Outcomes. CO-MTN includes implementation strategies to support the tiered care pathway including training and learning communities to support PCPs with improved knowledge and capacity and provide exceptional care for Colorado Long COVID patients, and practice facilitation to effectively implement the PCP care in their practice and coordinate with the MDCs. CO-MTN will help Long COVID patients in Colorado and have the potential for scaling to other states. To guide implementation and evaluation, we will utilize the Exploration, Preparation, Implementation and Sustainment dissemination and implementation framework. We will measure sustainment as continued involvement in the network during the final six months of the project period. Using qualitative, quantitative, and mixed methods, we will evaluate the Reach and Effectiveness for patients, and the Adoption, Implementation and Maintenance of the implementation strategies using the RE-AIM framework based on the following aims: Aim 1: Implement an integrated, tiered care delivery pathway with facilitated implementation support via CO-MTN and measure its effect on 1) Reach of Long COVID care to Colorado patients, specifically those described as underserved, and 2) Effectiveness on reducing Long COVID symptom burden and severity for patients engaged. Aim 2: Determine the effect of CO-MTN on 1) Adoption of the CO-MTN tiered care delivery model by primary care clinics and PCPs within these clinics, 2) Implementation of the tiered care pathway, and 3) Maintenance of the tiered care pathway key components past the active project period. Our team is comprised of national experts in Long COIVD care, practice-based research, and implementation science. Together, our multidisciplinary team will partner through CO-MTN structures and support to deliver high quality Long COVID care.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Considerable research has focused on drug use disorders as motivational disorders involving inherent or drug- induced reward pathway function. However, the focus of this application is on opposing aversive effects of methamphetamine (MA) that may curb its use, which have been little studied. The Richards (Phillips) laboratory identified the trace amine associated receptor 1 (TAAR1) as a critical target impacting MA-induced aversion. TAAR1 is an intracellularly located G protein-coupled receptor. MA gains access to TAAR1 only if it is transported into the cell. This occurs via extracellular membrane transporters, such as dopamine and serotonin transporters, DAT and SERT, respectively. We propose that TAAR1 activation by MA in dopamine and serotonin neurons is responsible for MA-induced aversion and that monoaminergic circuit interactions with the lateral habenula (LHb) are of particular importance. The overarching goal of the studies proposed in this application is to understand the monoaminergic neuron-LHb interactions responsible for the experience of MA- induced aversion via TAAR1 that may reduce risk for MA use. Our preliminary data show that MA activates lateral habenula (LHb) neurons, specifically in mice with functional TAAR1. The studies in Aim 1 will use slice electrophysiology to examine the TAAR1-dependent effects of MA in ventral tegmental area dopamine and dorsal raphe serotonin neurons, comparing slices from wildtype and CRISPRed knock-in mice, mice that have functional vs. nonfunctional TAAR1, respectively. Aim 1 studies will also use optogenetic stimulation to examine the effects of MA on glutamatergic synapses from the LHb. Based on our published findings, functional behavioral studies in this aim will examine the role of a glutamate receptor subunit, GluN2B, on MA aversion and intake. Aim 2 will perform behavioral studies focused on the LHb, which has been shown to mediate other types of aversion, has not been studied for MA aversion. We will ablate the LHb in mice with and without functional TAAR1 and study the impact on MA aversion and intake. Finally, Aim 3 studies will use a retrograde tracer to identify the LHb neurons that project to either the ventral tegmentum or dorsal raphe. Electrophysiological studies will determine whether dopamine or serotonin modulate MA activation of LHb neurons and determine whether MA activation of TAAR1 in presynaptic terminals of ventral tegmental area dopamine neurons or dorsal raphe serotonin neurons regulate the effects of MA using slices from mice with and without functional TAAR1. Thus, this proposal utilizes genetic tools, circuit analysis via electrophysiology and behavioral analysis to identify how MA engages LHb neurons in a TAAR1-dependent manner, whether LHb circuits are necessary for MA-induced aversion behaviors, and whether they inhibit MA intake. This strategy could be used to study effects of MA on TAAR1 signaling in other regions. The study of mechanisms underlying sensitivity to MA-induced aversion could lead to the identification of a new class of therapeutics.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Sepsis affects >70,000 U.S. children annually with 5-10% mortality, 35% long-term morbidity, and annual national costs of $7.3 billion. Outcomes are improved by timely diagnosis, which is less likely in general Emergency Departments (EDs) which treat adults and children. Although up to 80% of children with sepsis are treated in general EDs, the few studies in these settings have not identified strategies to improve diagnosis. Sepsis is an exemplar condition for challenges to timely diagnosis, highlighting diagnostic complexities noted in the National Academies of Sciences, Engineering, and Medicine’s 2015 report Improving Diagnosis in Health Care. This project also addresses challenges in pediatric emergency preparedness noted in the Institute of Medicine’s 2007 report, Growing Pains. The long-term goals of this work are to identify strategies to improve diagnosis of high-risk conditions in EDs. While establishing capacity to improve diagnosis across emergency conditions, the initial demonstration project will focus on pediatric sepsis, using methods of 1) identification, analysis, and reduction of diagnostic errors, and 2) work system improvements. It will uniquely leverage the transfer call center and the brief consultative phone call as an opportunity to apply diagnostic safety strategies and disseminate pediatric subspecialty knowledge. Phone calls with consultants are frequently used in diagnostic decision-making in EDs and have rarely been studied; this team will apply conversation analysis to recorded calls to identify opportunities to improve communication and the diagnostic process. This study will adapt and implement a diagnostic safety toolkit including: 1) a content-specific sepsis diagnostic checklist with demonstrated effectiveness in pediatric EDs, 2) improvement in subspecialty consultation processes to follow diagnostic safety principles. The study will be conducted in a children’s hospital transfer call center that receives calls about >18,000 children in >100 general EDs yearly. The aims are: SA1: Analyze the current diagnostic process in children with sepsis referred to the transfer center using mixed methods. Conversation analysis of recorded phone calls and quantitative analysis will guide evaluation of diagnostic accuracy and the diagnostic work system and process. SA2: Use the Implementation Mapping Adapt process to adapt diagnostic strategies (content-specific checklist and work system improvements) for implementation in the transfer center call process. SA3: Implement and evaluate the sepsis diagnostic safety toolkit in an 18-month implementation study. This work will establish strategies for improving the diagnosis of time-sensitive pediatric emergencies in general ED settings, where most children receive their critical first hours of treatment. Children’s hospitals’ transfer call centers are a unique, replicable opportunity to disseminate pediatric knowledge at the moment it is needed, improving diagnostic safety for children in any ED. Strategies identified in this work focused on content-specific checklists and work system improvements to the phone consultation process can be replicated in other conditions, improving diagnosis and safety in emergency medicine.

NIH Research Projects · FY 2025 · 2023-09

Proposal Summary There are no validated systems for identifying children without serious bacterial infection (SBI) upon admission to a pediatric ICU (PICU). Given the high prevalence of SBI among critically ill children (up to 46%) and risks associated with delayed antibiotic administration, nearly 50% of children without SBI receive antibiotics while microbiologic studies are pending. However, antibiotics can have adverse effects including acute kidney injury, clostridium difficile colitis, and development of antibiotic resistance. The long-term goal of this research is to validate and disseminate machine learning (ML)-based clinical decision support (CDS) tools able to improve PICU antibiotic decision-making thereby reducing antibiotic associated harm among critically ill children. In prior work, Dr. Martin developed ML-based predictive models, which use electronic health record (EHR) inputs (vital sign, laboratory, and other clinical data), to accurately identify children without SBI upon PICU admission in a single center retrospective cohort. The central hypothesis is that these models will demonstrate similar robust performance during prospective and multicenter evaluations, and that an antibiotic decisional needs analysis of PICU clinicians will inform the optimal design of model-based CDS tools. The central hypothesis will be tested via three aims: 1) prospectively evaluate two SBI predictive models within a single center EHR and determine the potential effect on antibiotic-days per child; 2) evaluate ML model generalizability by testing them in a multicenter EHR cohort; and 3) perform a multicenter, multidisciplinary antibiotic decisional needs analysis of PICU clinicians to facilitate user-centered design of equitable model-based CDS tools. In Aim 1, two SBI predictive models will be prospectively evaluated in silent fashion (predictions not revealed to clinicians) at a single center over two years. Model predictions will be compared to patient SBI outcomes to determine their negative predictive value and potential to reduce unnecessary antibiotics. In Aim 2, the same models will be applied to a retrospective dataset of six US children's hospital PICUs (~178,000 encounters over 8+ years) to assess generalizability by determining each model's negative predictive value and potential to reduce unnecessary antibiotics. In Aim 3, a rigorous qualitative content analysis of PICU clinician interviews from five institutions will identify the values driving antibiotic decision-making and enable user-centered design of model- based CDS tools. The research is innovative because it involves development of the first clinically validated system for excluding SBI at PICU admission and uses a ML approach to do so. The research is significant as it accelerates development of generalizable antibiotic decision-making tools to assist PICU clinicians in safely minimizing unnecessary antibiotics and associated harm. The educational component of this application will allow Dr. Martin to attain expertise in biostatistics, probability, ML bias, and study design, as well as technical skills in programming, ML, and CDS. This will allow him to transition to independence and make him uniquely qualified to develop, validate, and implement CDS tools able to improve the outcomes of critically ill children.