University Of Colorado Denver

NIH Research Projects · FY 2025 · 2021-04

Project Summary: Protein misfolding is implicated in many diseases, including cystic fibrosis, Alzheimer’s, Parkinson’s, and Huntington’s disease. Understanding protein folding mechanisms is therefore important for human health. While protein folding has been extensively studied for many years, the real-time folding of a protein has yet to be captured and quantified in a living cell due to a lack of adequate experimental spatiotemporal resolution. To fill the gap, I will combine single-molecule fluorescence microscopy and novel intrabodies that can distinguish unfolded and folded proteins to image cotranslational protein folding dynamics in living cells. With this technology, I propose to investigate the folding dynamics of the cystic fibrosis transmembrane conductance regulator (CFTR), the misfolding of which causes cystic fibrosis. CFTR is a good first application because it has already been demonstrated to predominantly fold cotranslationally in vitro. To characterize CFTR folding in living cells, I will develop genetically encodable intrabodies that bind folded and unfolded CFTR cytosolic domains (Aim 1). In parallel, I will establish methods to capture and quantify cotranslational protein folding dynamics using a model protein folding system based on GFP and its pre-existing intrabodies (Aim 2). With the technology from Aims 1 and 2 in hand (K99 phase), I will image CFTR cotranslational folding dynamics at the single mRNA level in living cells (Aim 3; R00 phase). This will reveal precisely when, where, and to what degree cotranslational CFTR folding is regulated within a fully natural context. Collectively, this work will not only shed new light on cotranslational protein folding dynamics, but will also lead to new strategies to combat cystic fibrosis and other protein misfolding diseases.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT: The long-term objective of this proposal is to understand genetic buffering, or how some individuals overcome the effects of a harmful genetic mutation. We generated a zebrafish model of buffering in order to understand how individuals who should have gotten sick are somehow able to live unaffected healthy lives. In our model, we used selective breeding to generate two strains of zebrafish that develop dramatically different head skeletons in response to the same harmful genetic mutation. In one strain, the mutation causes severe, lethal skeletal defects. Meanwhile, the other strain is remarkably buffered against the mutation. The buffered fish develop essentially normal head skeletons, surviving to be fertile, viable adults. We compare these strains to understand the natural buffering mechanisms that are present in some fish, and likely in some humans too. We hypothesize that buffering is due to factors that tune developmental processes to restore balance. For example, in Aim1 we will determine how DNA sequences which oppose the mutant gene can be turned down. In Aim 2 we examine how factors encoded in the DNA which perform the same function as the mutant gene can be turned up. In Aim 3 we will determine how changes that do not necessarily involve alterations in the DNA sequence can buffer the harmful mutation. These three specific aims test how interacting mechanisms function together to buffer development. We designed experiments to address these aims using state of the art methods like genome editing and sequencing, colorful cell and tissue labeling, quantitative measurements of large numbers of fish skeletons, and rigorous statistical analyses. The mechanisms we propose to study here are present in many developmental systems and organisms and therefore will likely be applicable in wide- ranging settings. This study of buffering mechanisms could lead to novel therapeutic approaches, buffering mechanisms might be manipulated in the future to manage disease symptoms. This work will also lead to a better understanding of the factors that make predicting genetic disease from gene sequences difficult. Thus, this line of research will potentially inform and improve medical practices, including genetic disease management, disease diagnosis, and counseling, falling squarely within the mission of the NIDCR to improve dental, oral and craniofacial health through research.

NIH Research Projects · FY 2025 · 2021-04

Plasticity in the hippocampus leads to persistent changes in synaptic structure and function that underlie learning and memory. Intracellular Ca2+ signaling pathways activated downstream of NMDA receptors (NMDAR) and L-type voltage-gated Ca2+ channels (LTCC) contribute to changes synaptic function that are required for initial expression of plasticity as well as changes in gene expression that support long-term maintenance of plasticity. In particular, activation of LTCCs plays a key role in dendritic spine structural plasticity and excitation-transcription (E-T) coupling to control the activity of transcription factors in the nucleus, such as cAMP/Ca2+-response element binding protein (CREB), nuclear factor of activated T-cells (NFAT), and myocyte enhancer factor 2 (MEF2). Alterations in LTCC function have been linked to multiple neurological and neuropsychiatric diseases. Importantly, NFAT-dependent transcription may control the expression of a number of target genes that play key roles in regulating E/I balance and excitability, including GABAA-Rs and voltage-gated potassium (Kv) channels. Our previous work established the scaffold protein AKAP79/150, which anchors the cAMP-dependent kinase PKA and the Ca2+-dependent phosphatase calcineurin (CaN) near LTCCs, as an essential regulator of E-T coupling via CaN-mediated dephosphorylation of NFAT. However, due to the large distances between synapses in dendrites and the nucleus in the soma, neurons face unique challenges in converting synaptic input into biochemical signals that control transcription. We recently found that LTP stimulated NMDAR-LTCC-NFAT synapse-to-nucleus signaling utilizes dendritic Ca2+ spike propagation to the soma as a novel E-T coupling mechanism. In addition, we found that this NMDAR-LTCC activation during LTP induction promotes Ca2+-induced Ca2+ release in dendrites that engages the endoplasmic reticulum (ER) Ca2+ sensor STIM1 to trigger negative-feedback regulation of LTCC Ca2+ influx while also mediating novel structural plasticity of the dendritic spine ER. However, there are still critical gaps in our knowledge regarding how NMDARs, LTCCs, and STIM1 operate over different spatial and temporal scales to control both local dendritic structural plasticity and distal dendrite-to-soma spike propagation to regulate transcription. Furthermore, we do not understand how the transcription of specific activity-regulated target genes is controlled by different patterns of activity transduced by these mechanisms to modulate key aspects of neuronal function, such as E/I balance. Thus, here we propose research to fill these gaps by characterizing the roles of postsynaptic LTCC Ca2+ signaling in mediating local structural plasticity in dendrites and Ca2+ spike relay from dendrites to soma (aim 1) in control gene of expression through NFAT and its co-regulators to impact E/I balance (aim 2).

NIH Research Projects · FY 2025 · 2021-03

Project Summary One of the major challenges in vaccine development is access to adjuvants that promote long-lived pathogen specific CD4+ and CD8+ T cell responses and provide protective cell mediated immunity. Our previous studies identified a molecular adjuvant that uses CD40 and TLR agonists (CD40/TLR) that induces protective long- lived T cell populations in mice and primates but why this approach is so effective is unclear. We believe that if we can understand why this adjuvant is different from other adjuvants or infection induced responses then it will help in the rational design of vaccines to generate protective cell mediated immunity. We have documented that vaccine-elicited T cells (Tvax) are distinct from infection-elicited T cells (Tinf) and this includes a unique metabolic program and unanticipated requirement for the cytokine IL-27 for Tvax generation. In addition, the ability of a specialized subset of dendritic cells (cDC1), to produce IL-27 predicts the magnitude of vaccine- elicited CD8+ T cell expansion and memory formation. Our recent studies have identified an IL-27-dependent c-Myc transcriptional signature within Tvax that is associated with T cell proliferation and survival. We developed a computational model which mathematically recapitulates T cell expansion data derived from antigen challenge studies in vivo. This unsupervised analysis indicates that the ability of this adjuvant to rapidly promote T cell interactions with APC at the initiation of T cell priming is the major predictor of the magnitude and quality of the T cell response. Based on these data sets will use intravital imaging of T–DC interactions and the manipulation of IL-27, DC functions and c-Myc pathways to understand the mechanistic determinants of the combined CD40L/TLR adjuvant. These data sets will be integrated into a stochastic agent-based mathematical model to predict and validate the key events involved in Tvax formation. The proposed studies bring together the combined efforts of three productive laboratories and their respective expertise in adjuvant discovery, CD8+ T cell biology, cytokine and transcriptional networks, multi photon imaging, and computational modeling in order to understand the molecular basis for adjuvant-elicited cellular immunity.

NIH Research Projects · FY 2025 · 2021-03

Project Summary With advancements in operative techniques and perioperative management, there is an increasing number of patients with single ventricle congenital heart disease (SV) that are surviving into childhood and beyond. Due to the chronic pressure and volume load placed on the single systemic ventricle, these patients remain at constant risk for the development and progression of cardiac failure. Unfortunately, very little is known about how the failing SV heart differs from the failing pediatric or adult biventricular heart. Additionally, the transition to heart failure that occurs in the SV heart is also incompletely understood. This lack of understanding in the mechanisms underlying SV heart failure are a major hurdle in the identification of effective targeted therapies. In addition, the rarity of SV makes it very difficult to perform prospective controlled drug studies as is routinely done in the adult heart failure population and as a result, treatments are based on extrapolation of clinical trials from different patient populations, anectdotal experience, or potential for theoretic perceived benefit. Phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are an example of such a therapy that is increasingly used in the SV patient population with a limited existing evidence-basis. Widespread, and fairly indiscriminate use of PDE5i for SV patients is driven in part by several publications suggesting positive clinical results in small series of SV patients. The recently published NHLBI FUEL (Fontan Udenafil Exercise Longitudinal assessment) trial demonstrated improved submaximal exercise in 400 fontan patients. These encouraging studies combined with our recent publication demonstrating increased PDE5 expression and activity in failing SV hearts suggesting that the myocardium may be a viable target of PDE5i. While historically the rationale for the use of PDE5i in SV is to augment pulmonary blood flow, we hypothesize that the failing SV myocardium, and specifically the mitochondria, represent a target of PDE5i therapy as well. Our preliminary data demonstrate: (1) Mitochondrial dysfunction, altered sirtuin signaling, and increased mitochondrial protein acetylation in failing SV myocardium (SVHF); (2) Decreased mitochondrial reactive oxygen species (ROS) generation detected by Electron Paramagnetic Resonance (EPR) in failing SV hearts treated ex vivo with PDE5i; (3) Decreased protein acetylation and improvement in mitochondrial function in failing SV hearts treated ex vivo with PDE5i; (4) Impaired mitochondria function in SV Non-Failing (SVNF) (primary transplant or Norwood specimens) hearts treated ex vivo with PDE5i; and (5) Mitochondrial dysfunction and increased ROS in primary cardiomyocytes treated with SVHF patient serum, which is improved by the addition of PDE5i or the SIRT 3 activator, honokiol (HNK). We hypothesize that mitochondrial dysfunction is involved in the HF transition of SV hearts, and that PDE5i improves mitochondrial function in failing SV hearts in a sirtuin-dependent manner. We propose the use of human tissue and a cardiomyocyte model to complete the proposed experiments. The purpose of this project is to understand the transition to HF in the SV population and provide pre-clinical evidence to inform more targeted use of, with the goal of optimizing clinical care and improving outcomes.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY Individuals living with Alzheimer's Disease and Related dementias (ADRD) may experience unwanted, intensive or burdensome end-of-life care because their preferences for care are unknown to their medical decision makers. For example, nearly 41% of individuals with ADRD undergo at least one intensive intervention (e.g., mechanical ventilation, artificial nutrition) in the last 3 months of life, which may prolong life but does not address quality of life. Thus, timely discussions about advance care planning (ACP) are imperative before individuals with dementia lose decision making capacity. Persons with cognitive impairment, spanning from mild cognitive impairment to dementia, are often cared for in primary care. However, primary care settings do not have effective models that can systematically integrate clinician-patient discussions and decision-making about ACP for older adults with and without cognitive impairment who still have decision making capacity. To address these gaps, we designed, refined with stakeholder input, and demonstrated the feasibility, acceptability, and efficacy of a theory-based and practical ACP group visit intervention, ENACT Group Visits (Engaging in Advance Care Planning Talks) among older primary care patients. ENACT Group Visits are two facilitated discussions, one month apart, led by a physician or advanced practice provider and a social worker. The intervention uses evidence-based ACP materials and leverages the group dynamic to promote patient goal-setting and self-efficacy. In a single-clinic pilot study, patients randomized to the ENACT Group Visits intervention had a 26% higher rate of ACP documents at 6 months, compared to a control arm of mailed ACP materials (p=0.007). This study aims to test the effectiveness of ENACT Group Visits to increase ACP documentation (one aspect of ACP) and to evaluate its effectiveness among patients across a spectrum of cognitive impairment, including early dementia. The proposed 2-arm, patient-level randomized trial will be conducted in five primary care clinics and is powered to compare ENACT Group Visits intervention vs control mailed ACP materials. We will use 1:1 allotment and patient-level block randomization by presence or absence of CI. The study will use components of PREPARE™, an evidence-based ACP program designed to decrease cognitive burden, and an easy-to-read advance directive. Aim 1 will determine the effectiveness of ENACT Group Visits vs control at 6 months to increase ACP documentation (primary outcome), ACP readiness, decision self-efficacy, and quality of communication (secondary outcomes). Aim 2 will determine whether the effectiveness of ENACT Group Visits intervention varies by cognitive impairment, including ADRD. Aim 3 will describe acceptability and feasibility, as well as intervention fidelity and implementation outcomes, of ENACT Group Visits intervention using qualitative and mixed methods. This primary care-based effectiveness trial of ENACT Group Visits intervention among older adults, including those with dementia, will generate needed evidence for implementing effective ACP interventions into real-world primary care settings.

NIH Research Projects · FY 2025 · 2021-03

PROJECT ABSTRACT The overarching goal of this Mentored Research Scientist Career Development Award (K01) is to provide Dr. Ruschelle Leone with the training and research activities needed to become an independent investigator. Her program of research will focus on developing and evaluating innovative integrated programs for heavy episodic drinking, cannabis use, and sexual assault. Research estimates 1 in 5 college women experiences sexual assault. Alcohol, cannabis, and sexual assault victimization have a reciprocal association; alcohol and cannabis are known risk factors for sexual assault, and women who experience sexual assault are at an increased risk of developing alcohol and cannabis use disorders. Although prevention efforts targeting potential perpetrators and bystanders is critical, these programs have yet to demonstrate reductions in rates of sexual assault victimization or perpetration. As part of comprehensive sexual assault programming, feminist scholars have called for the use of risk reduction programs to empower women by increasing a woman's ability to recognize and resist sexual assault by providing skills to avoid, interpret, and resist sexual assault. However, these programs have yet to directly target alcohol and cannabis use within an integrated framework. This innovative proposal includes training activities to ensure that Dr. Leone achieves the following four new career goals: 1) Develop expertise in reducing alcohol misuse, cannabis use, and sexual assault; 2) Receive training in the development and testing of integrated interventions for alcohol misuse, cannabis use, and sexual assault victimization; 3) Obtain advanced training in multilevel and longitudinal data analyses; and 4) Hone skills in grant-writing, responsible conduct of research, and professional development. The mentorship team includes expert psychologists, physicians, and public health scientists in the following areas: development of web-based interventions for alcohol and sexual assault (Gilmore), treatments for alcohol and cannabis use (Gray), social norm interventions for alcohol and cannabis prevention (Neighbors), sexual assault risk reduction programs (Senn), qualitative methods for intervention development (Salazar), design and analysis of clinical trials (Hayat), and program implementation (Self-Brown). Dr. Leone will apply the skills acquired during the training activities to a research project focused on the development of an integrated program for heavy episodic drinking, cannabis use, and sexual assault risk reduction. The research project includes mixed-methods research to develop program content, assess the usability and acceptability with college women who engaging in heavy episodic drinking and cannabis use, and conduct a feasibility randomized controlled trial. This project will establish feasibility of conducting a larger randomized clinical trial testing the efficacy of the integrated program. The proposed training and research activities will prepare Dr. Leone for an independent research career focused on the development and evaluation innovative integrated programs for heavy episodic drinking, cannabis use, and sexual assault. This project is directly in line with several NIH priorities and will inform a R-level grant to conduct a randomized clinical trial.

NIH Research Projects · FY 2025 · 2021-03

PROJECT Adverse morbidity when tissue associated maternal (FGT) likely SUMMARY pregnancy outcomes, including premature birth and stillbirth, are the leading causes of neonatal and mortality. A frequent cause of preterm birth and stillbirth is intrauterine infection, which occurs bacteria ascend from the vagina into the uterus and invade the amniotic cavity, leading to inflammation, damage, and adverse pregnancy outcomes. Group B Streptococcus (GBS) is one such bacterium with ascending infection and adverse pregnancy outcomes. The principal risk factor for this is vaginal colonization; however the mechanisms by which GBS persist i n the and ascend to the uterus remain unknown. GBS colonization status is intermittent and can be transient, reflecting a combination of GBS determinants, antagonism by commensal flora, and host immune , female genital tract responses. The current proposal seeks to address these dynamic aspects of GBS vaginal carriage, specifically 1) bacterial adherence to host cells/tissue of the FGT, 2) competition with vaginal microbiota, and 3) evasion of host defense. Recent studies have demonstrated that GBS stimulates vaginal epithelial exfoliation by activating epithelial-to-mesenchymal transition (EMT), leading to loss of barrier function and GBS dissemination to the upper FGT and fetus. We have recently discovered a GBS surface adhesin, BspC, that directly interacts with host intermediate filaments, including keratin 19 and vimentin, a canonical marker of EMT. We hypothesize that when EMT is induced the BspC-vimentin interaction plays an important role in GBS vaginal persistence and ascending infection. We have further discovered that GBS has a type VII secretion system (T7SS) that contributes to colonization. We hypothesize that T7SS is important for competition with vaginal microbiota for niche establishment and secreting anti-eukaryotic toxins that may invoke a host immune response. We have also demonstrated that IL-17A is produced during GBS colonization and that IL-17+ cells, such as MAITs and  T cells, actually contributed to GBS ascending spread. We hypothesize that IL-17 induced BspC-vimentin These hypotheses will be addressed in the following specific aims: AIM 1: Elucidate the contribution of BspC and intermediate filaments to GBS vaginal persistence, AIM 2: Examine the function of newly discovered GBS T7SS in mediating vaginal niche establishment and inter-bacterial competition, AIM 3: Determine the contribution of IL-17 and MAITs to the pathogenesis of GBS colonization. These studies should increase our understanding of the bacterial and host factors involved in the colonization and persistence within the FGT that impact GBS ascending infection and neonatal disease. IL-17 and producing T cells in the FGT induce EMT and barrier breakdown. This comes full circle; Once EMT is as a defensive response to initiate cellular exfoliation, GBS hijacks this process, possibly through a interaction, to persistent in the FGT.

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract The overarching goals of this proposal are to contribute to the understanding of the causes, mechanisms, and potential strategies for prevention of the international epidemic of chronickidney disease of unknown origin (CKDu). Our central hypothesis is that exposure to high concentrations of air contaminants will be associated with acute kidney injury in agricultural workers and that heat stress and dehydration will produce adverse effects on kidney biomarkers. We further hypothesize that this damage occurs through a vasopressin-mediated injury pathway. Access to a population of sugarcane workers in cooperation with a major Guatemalan agribusiness will allow for a study that characterizes sugarcane worker exposure to particulate matter (PM) and it constituents (silica, glyphosate, and metals) in conjunction with personal factors, including dehydration and heat stress. We will examine the individual contribution of air contaminant exposure, as well as the combined contribution of exposure and personal risk factors, on kidney dysfunction. In addition, we will investigate the mechanistic role of the vasopressin pathway to pathogenesis of CKDu. This exposure pathway development relationship dehydration investigate an unexplored identify may workers at risk for the of kidney dysfunction by conducting a robust personal exposure assessment, b) evaluate the between exposure(s) and acute kidney injury, and c) examine underlying mechanisms by which and heat stress contribute to increased risk of CKDu in conjunction with nephrotoxicant exposures. research will: a) to inhalation exposures that place agricultural To metals, on evaluate identify address this hypothesis, three aims are proposed: Aim 1 focuses on characterizing exposure to PM, silica, and glyphosate in Guatemalan sugarcane workers and examining the impact of meteorological factors personal exposure. We will use prospective, quantitative personal measurements in workers. Aim 2 will the relationship between occupational air contaminant exposure and kidney biomarkers of effect to workers withincreased risk of cross-shift worsening renal function and inflammation. Aim 3 will evaluate the potential mechanism by which environmental and individual risk factors induce kidney injury. The proposed panel study will collect repeat respirable PM personal air samples across two 6-month harvest seasons in two groups of 60 clinical Guatemala. data and Next, biological workers, totaling 120 workers samples from participants at . First, the we will collect baseline questionnaire and time of hire at a sugarcane plantation in we will collect personal air measurements for each participant during the entire work shift to estimate daily airborne exposure glyphosate, and heavy metals, as well as urine and blood biomarkers pre- and post-shift t three time points for each worker during the two seasons. to silica, a Resultsof this research will lead tolarge-scale intervention trials that will help to prevent CKDu by targeting potential therapeutic approaches for vulnerable populations that can be disseminated internationally.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT Hemophilia is an inherited deficiency of clotting factor VIII or IX, which causes joint bleeding and crippling arthropathy, along with muscle bleeding. Prophylactic factor medication administered intravenously several times per week decreases but does not eliminate bleeding. Most efforts to eliminate breakthrough bleeding on prophylaxis alter medication regimens to increase factor levels; however, this is often not successful and comes with increased financial burden and/or more frequent injections. The effort to eliminate breakthrough bleeding is limited by an incomplete understanding of bleeding biomechanics. This knowledge gap also constrains choices for safe and effective physical activity, which is important in persons with hemophilia (PwH) to prevent obesity, improve social functioning, and decrease mental health concerns. This study develops methods to objectively measure forces related to lower extremity joint and muscle bleeding in PwH, in order to fulfill the long-term goal of decreasing bleeding risk. This long-term goal will be accomplished in a future study that will collect data to build a time-dependent statistical model of bleeding risk that includes hemostatic, joint fragility, and biomechanical bleeding risk components. The objectives of the current study are to determine how past joint bleeding influences asymmetry in lower extremity joint loading, measured in a Motion Lab using motion capture and force plate technology (aim 1); to determine the influence of motion lab asymmetry on future lower extremity joint and muscle bleeding (aim 2); and to develop tools to collect motion data outside the Motion Lab using inertial measurement units (IMUs) (aim 3). The immediate goals of the candidate for this K23 award, Dr. Beth Warren, are to add new skills to her bioengineering and hematology background. These include motion measurement techniques inside and outside the Motion Lab, including signal processing techniques; hemophilia joint outcome measurement and meaning; and statistical model planning for future studies. Achieving these goals will support her long-term career goal to become a leading expert in hemophilia joint outcomes and activity-associated bleeding risk. Dr. Warren will accomplish her research and training aims with the support of her mentors, Dr. Marilyn Manco-Johnson (hemophilia clinical research) and Dr. James Carollo (biomechanics, movement analysis). The University of Colorado Hemophilia and Thrombosis Center (UC-HTC) will provide world-class infrastructure to support Dr. Warren’s career development and research objectives, including facilities and research staff with extensive experience and expertise in hemophilia clinical research. The University of Colorado provides junior faculty members with numerous opportunities to facilitate the transition to research career independence. By completing her research and training aims, Dr. Warren will advance the science of objective measurement of bleeding biomechanics, while also obtaining experience and training necessary to begin her career as an independent investigator.

NIH Research Projects · FY 2025 · 2021-02

Project summary Patients with Alzheimer’s disease (AD) are known to have greater balance and gait impairment and double the rate of falls relative to healthy older adults (60-70% vs ~30%). Currently, few effective interventions exist to manage and mitigate falls in AD, and unfortunately, falls continue to be one of the primary drivers of morbidity, institutionalization, and mortality among AD patients. Recent studies have shown that AD patients have a two-fold higher prevalence of vestibular impairment (~50%) relative to age-matched controls (~25% prevalence). Additionally, in pilot data from an ongoing observational study, we have shown that vestibular loss is associated with an increased rate of falls in AD patients. Vestibular therapy (VT) is a well-established treatment for vestibular loss, and consists of physical therapy-based exercises designed to foster compensation for reduced vestibular function. VT is effective in improving balance and reducing fall risk in cognitively-intact patients with vestibular impairment. However, whether VT could be effective in improving balance and reducing falls in AD patients with vestibular loss has never been explored. In this study, we propose a randomized controlled trial of VT in 100 patients with mild-moderate AD who have vestibular impairment. We will randomize patients 1:1 to a standard course of VT (1 session per week for 8 weeks) or to an active control matched for effort and duration. We will compare the efficacy of VT vs. active control primarily on 1-year incident fall rate. We will also investigate potential intermediate outcomes between VT and falls – specifically balance outcomes (a predicted target of VT) and spatial cognitive outcomes (a novel target of VT) – to understand potential mechanisms by which VT may influence fall rates. We hypothesize that VT will have preliminary efficacy in reducing 1-year incident fall rates relative to an active control intervention in a convenience sample of 100 patients with mild-moderate AD. We will also explore whether VT has preliminary efficacy in improving balance and cognition relative to the active control intervention. To accomplish the proposed study, we will recruit 100 patients from the Memory and Alzheimer’s Treatment Center, a well-established AD clinical research resource at Johns Hopkins. Falls are a disastrous outcome in patients with AD. If this pilot trial followed by a Phase III multi-center trial provide strong evidence for the benefit of VT in reducing falls, this low-risk intervention could be widely disseminated and implemented by an existing workforce and infrastructure, and produce substantial, sustained change in AD clinical practice.

NIH Research Projects · FY 2025 · 2021-02

Pathogenic variants in the Cyclin-dependent kinase like 5 (CDKL5) gene cause CDKL5 deficiency disorder (CDD, MIM 300672, 105830) a severe developmental and epileptic encephalopathy (DEE) associated with cognitive and motor dysfunction and cortical visual impairment. Recent data suggest CDD is one of the most common genetic causes of DEE. Work in CDD animal models has demonstrated the ability for disease modification and symptom reversal: worldwide efforts are now underway to develop therapeutic strategies (including gene therapy) to treat and potentially cure CDD. While there are four active clinical trials, none assesses the full spectrum of this DEE to address true disease modification. While capability for disease modifying therapies is accelerating, there is a critical barrier for clinical trial readiness that may result in failure of these therapies, not due to lack of efficacy but due to lack of validated outcome measures. CDD has been associated historically with Rett syndrome but there are many clear distinctions and CDD has emerged as an independent disorder. Some Clinical Outcome Measures (COMs) can be adapted from Rett syndrome COMs, whereas others need to be developed specifically for CDD. Our research network is uniquely positioned to develop clinical trial readiness for CDD by pairing exceptional experience in the development and validation of outcome measures with an extensive network of CDD experts and clinical trialists. Our goals are to 1) refine and validate appropriate quantitative COMs and biomarkers and 2) conduct a multi-site clinical trial readiness study to ensure that they can be successfully implemented. We will test the hypothesis that CDD specific COMs can be refined to accurately and reproducibly track meaningful changes in clinical trials: Aim 1: Generate and validate a suite of COMs and biomarkers necessary to comprehensively assess disease modification in CDD. Aim 2: Conduct a multi-site clinical trial readiness study to assess implementation, longitudinal stability, and collect baseline COMs and EEG/evoked potential data. Overall Impact: These outcome measures will establish clinical trial readiness for CDD and generate historic baseline outcome data, ensuring optimal testing of potential new therapeutics including gene therapy. Furthermore, these measures will be adaptable to other DEEs by enabling choices of outcome measures beyond existing NINDS supported measurement tools (NeuroQoL, PROMIS, Toolbox) that are not designed for the severity of the DEE populations.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract Cancer screening for the right patients at the right level of cancer risk can save lives, but over the last several years many expert groups have de-intensified mammography screening recommendations because of evidence of net harm for certain populations. Newer recommendations for mammography screening highlight a tailored approach based on age and risk and comorbidities, aiming to minimize harms and reach women who could benefit the most. For example, according to the United States Preventive Services Task Force guidelines, women age 40-49 should talk with their doctor and make an informed choice about whether to initiate screening. The change in emphasis from strongly promoting annual mammograms for all women over 40 to promoting informed choice in this age group constitutes a medical reversal, in the sense that this new message is very different from past messages and people’s expectations. Moreover, the recommendation for informed choice can elicit negative reactions in some women. Many women express disbelief when told about overdiagnosis and overtreatment, which are significant harms of screening. The notion of risk-based screening—that is, creating a screening plan tailored to a woman’s objective cancer risk—can raise suspicions of healthcare rationing. The result is a delicate situation in which there is need to convey the evidence to women so they can make an informed choice, but also a need to do it in a way that maintains credibility and trust despite this health message reversal. In this research we focus on four types of concerning responses that women may express in reaction to mammography evidence: Reactance (i.e. perceived manipulation or influence, e.g. “this is trying to ration healthcare”), self-Exemption (e.g., “this doesn’t apply to me”), Disbelief (e.g., “you can’t believe all the research anyway”), and Source derogation (e.g., “I don’t trust this source”), which we shorten to REDS. This research will identify affective and cognitive predictors of these reactions, and identify the consequences of these reactions for screening preferences and shared decision-making. In Aim 1 we will conduct a nationally representative survey of women age 40-49 and test affective and cognitive predictors of REDS reactions and consequences for screening intentions. In Aim 2 we will conduct a longitudinal survey and identify how women’s attitudes toward the evidence predicts shared decision making for mammography in an upcoming primary care appointment, and we will also examine how these attitudes change over time and are influence by women’s broader social environment. In Aim 3, we will conduct interviews and focus groups to identify strategies for communicating more effectively about screening, to avoid negative REDS responses, and promote positive responses like empowerment and desire for shared decision making. We will modify an existing mammography decision aid to incorporate these improvements, and then conduct randomized pilot tests of these improvements to provide preliminary evidence that they reduce REDS and improve women’s positive responses to mammography evidence.

NIH Research Projects · FY 2026 · 2021-02

PROJECT ABSTRACT Alzheimer’s disease (AD) treatments designed to target the amyloid-beta peptide have shown encouraging results in transgenic animal models but less encouraging results in human trials, which have also been plagued with serious adverse events (SAEs), including amyloid-related imaging abnormalities (ARIAs). Our proposed innovative therapeutic approach is based on epidemiological evidence that patients with the inflammatory disease rheumatoid arthritis (RA) have a reduced risk of developing AD, unrelated to their use of non-steroidal anti-inflammatory drugs (NSAIDs). We identified the innate immune system stimulant Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as a hematopoietic factor upregulated in RA, which we found reduced brain amyloidosis and reversed cognitive impairment in transgenic AD mice. Other studies have shown GM-CSF to be neuroprotective, anti-apoptotic, and neurogenic in several models of neurological diseases and injuries. We also found that recombinant human GM-CSF(sargramostim/Leukine) treatment is associated with cognitive improvements in leukemia patients after bone marrow chemo-ablation and hematopoietic cell transplant therapy. Notably, sargramostim is an FDA-approved drug for increasing the production and differentiation of white blood cells with an excellent safety record over 30 years. Most importantly, we recently completed a Phase I/II safety and efficacy trial (NCT01409915) in which mild-to-moderate AD participants were treated with sargramostim (250 mcg/m2/day SC) or placebo five days/week for three weeks (20:20 participants per group) with neurological, neuropsychological, neuroimaging, and blood biomarker assessments. Sargramostim treatment was safe (Primary Endpoint) with no drug-related SAEs and no ARIAs. Furthermore, the Mini-Mental State Exam (MMSE) showed cognitive improvement in the sargramostim group at the end of treatment (EOT) compared to baseline (p=0.0074) and in the sargramostim group compared to the placebo group at the EOT (p=0.037) and at 45 days after the EOT (p=0.0281). Other assessments showed no treatment benefits, but there was a trend negative correlation between changes in MMSE versus amyloid-PET. We now propose to carry out a randomized, double- blind, placebo-controlled trial in 42 mild-to-moderate AD participants, 28 of whom will receive sargramostim (250 mcg/m2/day SC) and 14 of whom will receive placebo, five days/week for 24 weeks with a 45-day follow-up visit. We have received both an IND exemption (134291) and IRB approval (17-0215) but will submit improved versions in the coming months. Our Specific Aims are: 1) Assess the long-term safety and tolerability of sargramostim in mild-to-moderate AD participants (Primary Endpoint). 2) Assess the effects of sargramostim treatment on cognition and activities of daily living in mild-to-moderate AD participants (Secondary and Exploratory Endpoints). 3) Assess changes in biomarkers associated with sargramostim treatment in mild-to- moderate AD participants (Exploratory Endpoints).

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central mediator of two opposing forms of NMDA- receptor (NMDAR)-dependent synaptic plasticity: long-term potentiation (LTP) and depression (LTD). Pathological overstimulation of NMDARs during cerebral ischemia causes excitotoxic neuronal cell death, and we have recently shown that CaMKII mediates also the neuronal damage after global cerebral ischemia (GCI). Importantly, in vivo injection of our optimized CaMKII inhibitor (tatCN19o) provided significant neuroprotection after GCI models that closely mimic the most relevant human conditions: cardiopulmonary resuscitation (CPR) after cardiac arrest in mice or after ventricular fibrillations in pig (unpublished). CaMKII inhibition (i) was done at a highly clinically relevant timepoint for these conditions (30 min after CPR); (ii) was effective also in conjunction with current standard of care (therapeutic hypothermia); and (iii) protected not only from neuronal cell death but also from the long-lasting functional impairments in LTP that are seen in the surviving neurons. Here, three connected but independent aims will directly promote, our mechanistic understanding of CaMKII- mediated regulation of neuronal cell death and LTP impairment. Specifically, the project will investigate (1) the cross-talk of CaMKII autonomy mechanisms in mediating ischemia-induced neuronal damage, (2) a possible dual role of CaMKII in neuronal cell death versus survival, and (3) mechanisms that underly the CaMKII- dependent long-term LTP impairment of the neurons that survive after ischemia. Together, the results of this study will significantly advance our understanding of the molecular mechanisms underlying ischemic neuronal cell death. Additionally, they will inform future development of a therapy in humans.

NIH Research Projects · FY 2024 · 2021-01

PROJECT SUMMARY. Despite knowing for more than 60 years that three copies of chromosome 21 (chr21, T21) is the genetic cause of Down syndrome (DS), we still know relatively little about how T21 drives the vast majority of DS pathophysiology. Over the last several decades, alterations in the response to diverse environmental stress conditions, such as oxidative stress, the unfolded protein response, the integrated stress response, and immune signaling, have been identified in T21 cells and mouse models of DS. Indeed, we have shown that the interferon (IFN) response is constitutively active in T21 cells, leading to an interferonopathy-like state of global immune dysregulation, likely due to the presence of four IFN receptors (IFNRs) in a single locus on chr21. Recently, we identified the IFNR locus as a target of directed transient site-specific gene amplification (TSSG) in response to viral infection. Importantly, we found that while the IFNR cluster is subject to TSSG in healthy cells, this process is impaired in T21 cells. These TSSGs arise from DNA rereplication events that are normally regulated by epigenetic changes to chromatin structure. Our prior work determined that DNA rereplication and TSSG are part of cellular responses to myriad environmental stress, including hypoxia, UPR, and heavy metal exposure, implicating dysregulation of DNA rereplication as a potential common element in the altered stress responses of T21 cells. This project will test the transformative hypothesis that dysregulated DNA rereplication in response to cellular stress contributes to the development of T21 and subsequent pathophysiology of DS. The potential impact of this hypothesis is profound. Not only could the dysregulation of IFNR TSSG affect the response to immune activation and pathogen response, it is entirely possible that other stress response- mediated TSSGs are also impaired, with subsequent effects on systemic biology. Furthermore, recent studies have shown that extrachromosomal DNA can lead to missegregation of chromosomes, leading to our hypothesis that virally-induced DNA rereplication of the IFNR locus during gamete development could promote missegregation of chr21 leading to T21. Given the novelty of our observation and hypothesis, this proposal seeks to answer a number of questions regarding TSSG in T21 including: 1) How do T21 cells suppress TSSG of the IFNR locus? 2) Do T21 cells fail to initiate rereplication at other stress-induced TSSGs? 3) Do T21 cells have a different DNA rereplication program? and 4) How might amplification of the IFNR locus contribute to missegregation events that lead to T21? Using a combination of cell culture models from humans and mice and a novel mouse in vitro fertilization model, we will comprehensively determine the extent of dysregulation of DNA rereplication in T21 and how DNA rereplication may contribute to the development of T21 and pathophysiology of DS.

NIH Research Projects · FY 2025 · 2021-01

Project Summary Acute respiratory distress syndrome (ARDS) is a rapid onset respiratory failure that is caused by factors ranging from pneumonia to sepsis. The impact of ARDS is substantial with more than 200,000 cases per year in the United States and an estimated mortality rate of 40%. All ARDS patients are mechanically ventilated to overcome the derangements in lung function caused by pulmonary edema, surfactant inactivation, and alveolar collapse. However, this essential mechanical ventilation can cause additional ventilator-induced lung injured (VILI) through tissue overdistension (volutrauma), the cyclic collapse and reopening of small airways and alveoli (atelectrauma), and inflammatory effects (biotrauma). Since VILI is a risk in all ARDS patients, and a significant contributor to ARDS mortality, improvements in ventilatory management are a key step in improving ARDS survival. However, further refinement of ventilation protocols to reduce VILI is challenging because of differences between patients and the changes in lung function that occur over time as ARDS worsens or resolves. Because of this inter- and intra-patient variability, ventilation that is beneficial in one person can be harmful in another. To overcome this challenge, we postulate that ventilation should be guided using a VILI cost function that provides real-time feedback of ventilation safety by describing the amount of VILI that is occurring. Our study will define such VILI cost functions based on the changes in lung function, structure, and inflammation that are the result of injurious ventilation. Using the cost function as a guide, the optimally safe ventilation for each patient could be determined by manually adjusting the ventilator settings. However, given the large number of permutations of ventilation adjustments this is not a practical approach. Instead, we will develop a mathematical model to predict optimal ventilation for each patient. These simulations will be personalized by fitting to real time pressure-flow measurements and then used to find the ventilation pattern that minimizes the VILI Cost Function. The predicted optimally safe ventilation will then be applied, and the process repeated to account for changes in lung function over time. The potential benefits of the proposed study are substantial. The VILI cost functions we define will provide an essential measurement of ventilation safety. Our innovative approach to optimize lung-protective ventilation using predictive models may lead to decreased ARDS mortality by protecting the injured lung while, at the same time, reducing provider workload. The proposed system also represents a paradigm shift in the way that ventilation strategies are established. Instead of testing a strategy in animal models and then in the heterogeneous ARDS patient population, where the effect may be beneficial to some patients and harmful to others, focus may be directed towards identifying algorithms that predict and prevent VILI independent of ARDS phenotype and lung mechanical function.

NIH Research Projects · FY 2025 · 2020-12

SUMMARY/ABSTRACT This proposal requests supplemental funding to support the ongoing NIH/NIDDK-funded R01 project (DK123334), which was initiated in December 2020 for a five-year period. The parent study aims to compare the 4-week effects of frequent, short bouts of physical activity (PA) designed to interrupt prolonged sedentary behavior (SB, i.e., sitting) versus a single continuous daily PA bout of equal total duration, focusing on their impact on glucose control and the underlying mechanisms in adults with prediabetes. The PI, a mid-career researcher, faced significant challenges during the initial years of this R01, including launching a new clinical research study and assembling a team during the COVID-19 pandemic, as well as navigating the demands of new parenthood following the birth of her first child in Spring 2022. These factors inevitably impacted her work productivity. Despite these challenges, the study has successfully randomized 35 out of the planned 66 participants, with primary outcome data collection completed for 32 participants. Additional pilot grants have enabled the extension of the intervention from 4 weeks to 12 weeks, along with a 3-month follow-up and expanded data collection. With over 60% of the data now collected, the focus is shifting to the critical tasks of creating a central database, data cleaning, and analysis to accelerate the generation of interpretable preliminary data. The supplemental funding is sought to hire a new PRA with skills and experience in database management, data cleaning, coding, and analyses. Three specific aims will be addressed. Aim 1 involves the analysis and interpretation of daily PA and SB data, along with 24-hour glycemic variability, including an exploratory analysis to better understand the interaction of these factors in free-living conditions. Aim 2 focuses on the analysis of 24-hour substrate oxidation and glucose kinetics using the combination of whole-room calorimetry and a dual- tracer technique to explore how different PA patterns influence glucose metabolism. Aim 3 proposes to explore whether distinct glycemic phenotypes are associated with changes in traditional markers of glucose metabolism following the interventions, potentially leading to personalized strategies for T2D prevention. This funding is crucial for accelerating data analysis, facilitating the timely dissemination of findings, and generating the preliminary data necessary to develop a new, competitive R01 proposal. It will also give the PI the time needed to focus on manuscript preparation and grant writing, thereby advancing her project and ensuring her retention and success in academia. Ultimately, these data will contribute to scientific and public health efforts aimed at optimizing PA interventions for T2D prevention.

NIH Research Projects · FY 2025 · 2020-12

Project Summary Autism Spectrum Disorders (ASDs) comprise a group of severe neurodevelopmental disorders that are typified by communication deficits and social impairment. Given that the onset of symptoms occurs by the age of 3, it is largely agreed that neuronal dysfunction arises during early brain development. A developing brain shows a remarkable capacity for plastic changes in response to experiences; thus, its development is most vulnerable to the environmental factors that can derail normal brain function. In utero exposure to drugs that raise blood 5HT levels, including selective serotonin reuptake inhibitors (SSRIs), has demonstrated behavioral and psychological deficits in offspring that closely resemble autistic symptoms in both animals models and human studies. In order to understand how these alterations arise, it is necessary to first understand the basic mechanisms of serotonergic modulation of brain function. Formation and stabilization of excitatory synapses are known to be essential for the initial establishment of functional neural circuits. Conversely, disrupted synapse development impairs neuron function and is thought to underlie the pathology of multiple neurodevelopmental disorders. PFC is densely innervated by serotonergic axon terminals and associated with higher cognitive processes that may be disrupted in illnesses such as ASDs. Despite a wealth of literature examining the role of 5HT in modulating behavior and in the pathogenesis of brain disorders, little is known at the cellular and molecular level about the role of 5HT in early cortical development, and particularly the postsynaptic 5HT mechanisms that modulate synapse development in the developing PFC. In the present study, we utilize a novel combination of tools including two-color, two-photon uncaging that enables precise release of 5HT and glutamate neurotransmitters, calcium imaging, electrophysiology, and optogenetic stimulation of genetically-targeted 5HT neurons to test our central hypothesis that 5HT signaling promotes the initiation of excitatory synapse formation and controls the maturation of excitatory synapses during brain development. Guided by strong preliminary data, we will examine this hypothesis in two specific aims: 1) Determine the role of 5HT signaling in lowering the threshold for induction of activity-dependent synapse formation. 2) Define the actions of 5HT on activity-dependent, input-specific and heterosynaptic spine stabilization. Results from these studies will further our understanding of the unique and detailed mechanisms by which 5HT regulates brain development, with critical relevance to cellular underpinnings of neurodevelopmental disorders. In the U.S., approximately 13% of pregnant women use SSRIs, which typically increase fetal 5HT levels. We expect that our results will highlight new avenues into the investigation of the pathophysiology underlying neurodevelopmental disorders resulting from early perturbation of 5HT signaling.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY People with diabetes are at high risk of developing a range of diverse microvascular, macrovascular and neuropathic complications that are associated with high morbidity and erode their quality of life. Diabetes is expected to take an increasingly large financial toll in in the United States (U.S.) the future, particularly among working age adults. Ongoing, timely and efficient surveillance of type 1 and type 2 diabetes diagnosed among young adults aged 18-45 years is essential to identify health disparities and inform health care systems and the public health community to identify and prioritize strategies to prevent diabetes and its complications. The Colorado DiCAYA surveillance team is ideally situated to address the critical challenge of utilizing existing electronic data sources to generate accurate, timely estimates of the incidence and prevalence of diabetes among the young adult population by type, age, sex, and race/ethnicity subgroups. In response to RFA-DP-20- 001- Component B, we propose to ascertain the annual prevalence and incidence of diabetes among adults aged 18-45 years of age in the state of Colorado starting with year 2020 from the following data sources: 1) electronic health records from the well-established SEARCH network of endocrinology clinics, community clinics and hospital networks in Colorado; 2) a state-wide, legislatively mandated All Payers Claims Database that contains over 33 commercial health plans and 100% of Medicaid claims; and 3) the University of Colorado Health System data warehouse (Health Data Compass). Our simple, yet innovative integrated surveillance approach will utilize a combination of algorithms, incremental record linkage and targeted chart review to identify young adults with diabetes, distinguish diabetes type and estimate date of diagnosis. Our specific aims are: Aim 1: SURVEILLANCE (Prevalence)- To ascertain cases of prevalent diabetes among young adults age 18-45 years, by age, race/ethnicity and diabetes type; Aim 2: SURVEILLANCE (Incidence)- To ascertain newly diagnosed diabetes cases in young adults age 18-45 years at diagnosis, by age, race and diabetes type; Aim 3: EVALUATE PUBLIC HEALTH SURVEILLANCE METHODS - To evaluate the strengths and challenges of our integrated surveillance approach to determine the burden and incidence of diabetes among young adults 18-45 years by ascertaining validity, completeness and representativeness of case ascertainment methods.

NIH Research Projects · FY 2024 · 2020-09

Project Abstract We propose to develop a chronically implantable, all optical, optogenetic nerve interface that can non- invasively, optically neuromodulate individual axons of nerves in the parasympathetic or peripheral nervous system. The proposed interface would benefit treatment of human disease and disabilities related to the thoracic and abdominal organs and systems innervated by the cervical vagus nerve, such as epilepsy and metabolic disorders. We propose to optically interface from afferent/efferent axons in these nerves with the goal of modulating organs or brain circuits innervated by them. The bidirectional optical neural interface technology will utilize the capabilities of optogenetics enabled through viral vector transfection of afferent and/or efferent neurons with genetically targeted, optically activated reporter proteins and opsins. Our central premise is that we can use optics to communicate with axons in a nerve. For optical approaches to work we need to convert action potentials into an optical signal. This can be done using genetically encoded calcium indicators or other voltage sensitive proteins that change their fluorescent properties upon action potential generation in a neuron. Because nerves do not naturally express optical proteins, we will work with transgenic mice that express these proteins and use these mice models to refine our system before making it available for other researchers to use. We aim to develop a compact, bench-top optical system that can be shared with other research labs to provide the unique ability of being able to interrogate specific fascicles and axons within the nerve. In the future, this technology has potential for translation to human clinical applications. The technology in the proposal is ambitious, but we have formed an outstanding team of cell biologists, neuroscientists, biomedical, electrical, and mechanical engineers. The team has an excellent track record of successful collaborations on multiple grants and publications.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY: Significant gaps existed at the end of the 20th century in our understanding of the incidence and burden of diabetes in youth. The SEARCH for Diabetes in Youth, initiated in 2000, has taught us that diabetes among youth is frequent and increasing in the U.S. The prevalence of type 1 diabetes (T1D) and type 2 diabetes (T2D) among youth <20 years of age increased by 21% and 30.5%, respectively between 2001 and 2009. The increasing burden of T1D is seen in most racial/ethnic and age groups while increasing trends in the burden on T2D are noted in 10-14 and 15-19 year old Hispanic, non-Hispanic white and non- Hispanic black youth. Similarly, the incidence of both T1 and T2D is increasing in the U.S (1.4% and 4.2% annually between 2002-2012, respectively). T1D is no longer a rarity among minority youth, with the steepest trend in incidence observed among Hispanic youths. The incidence of T2D has increasing among all race/ethnic groups except non-Hispanic whites. Ongoing, timely and efficient surveillance of diabetes diagnosed in youth is essential to identify health disparities and inform health care systems and the public health community to identify and prioritize strategies to prevent diabetes and its complications. While the SEARCH surveillance approach clarified many gaps in our understanding of diabetes among youth, less costly, time- and labor-intensive approaches are needed that utilize the enormous wealth of data in the electronic medical record and other clinical datasets, while maintaining the critical infrastructure and expertise developed during SEARCH. The Colorado DiCAYA surveillance team is ideally situated to address the critical challenge of utilizing existing electronic data sources to generate accurate, timely estimates of the incidence and prevalence of diabetes among youth, by age, sex, and race/ethnicity subgroups. In response to RFA-DP-20-001- Component A, we propose to ascertain the annual prevalence and incidence of diabetes among youth <18 years of age in the state of Colorado starting with year 2020 from the well-established SEARCH, network of pediatric endocrinology clinics, community clinics and hospital networks in Colorado. Our simple, yet innovative integrated surveillance approach will utilize a combination of algorithms and targeted chart review to identify youth with diabetes, distinguish diabetes type and estimate onset date. Our specific aims are: Aim 1: SURVEILLANCE (Prevalence)- To ascertain cases of prevalent diabetes among youth age < 18 years, by age, race/ethnicity and diabetes type ; Aim 2: SURVEILLANCE (Incidence)- To ascertain newly diagnosed diabetes cases in youth age < 18 years at diagnosis, by age, race and diabetes type; Aim 3: EVALUATE PUBLIC HEALTH SURVEILLANCE METHODS - To evaluate the strengths and challenges of our integrated surveillance approach to determine the burden and incidence of diabetes among youth < 18 years by ascertaining validity, completeness and representativeness of case ascertainment methods.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Our long term goal is to reduce postoperative infections. We will start by developing a system to accurately and completely identify their occurrence by applying machine learning algorithms to electronic health record (EHR) data. We will utilize a comprehensive audit and feedback system to create reports of risk-adjusted rates and specific details of postoperative infectious complications that are shared with surgeons and other healthcare providers to facilitate their awareness. We call this system the Automated Surveillance of Postoperative Infections (ASPIN). ASPIN will be piloted in the four major hospitals of the University of Colorado Health system (UCHealth) with a combined surgical volume of approximately 80,000 patients per year. We expect this will supersede the costly and laborious manual partial sampling of postoperative infectious complications which is current utilized by many hospitals. Specific Aim 1. Expand and enhance models for preoperative risk prediction and postoperative identification of surgical infections using EHR and ACS NSQIP data from patients who underwent operations at four UCHealth hospitals. Specific Aim 1a) Enhance previously-developed models for identification of postoperative infections by controlling Type-I errors via “knockoffs,” a recent statistical innovation for high dimensional model selection using false discovery rate correction. Specific Aim 1b) Deploy natural language processing methods using EHR text reports of these patients to identify additional indicators of postoperative infections and further refine the models. Specific Aim 1c) Create preoperative risk models for infection using EHR data - similar to the models implemented in the AHRQ-funded Surgical Risk Preoperative Assessment System - but that do not require additional data entry by the health care providers. Specific Aim 2. From the beginning of the study, develop ASPIN with input from an Advisory Committee composed of administrators and surgeons from all four UCHealth hospitals. Additional feedback from surgeons will be obtained through focus groups and semi-structured interviews at several steps of ASPIN development and implementation planning. Specific Aim 3. A pilot implementation of ASPIN will utilize the RE-AIM framework to guide and examine the preliminary effectiveness and feasibility of ASPIN at UCHealth. We will recruit 30 surgeon participants from all four UCHealth hospitals to use ASPIN, and we will evaluate the reach, effectiveness, adoption, and implementation of ASPIN. This research responds to AHRQ priorities by utilizing existing data to develop a learning health system with a distinct focus on improving surveillance and reporting of postoperative healthcare-associated infections.

NIH Research Projects · FY 2024 · 2020-09

Abstract Antiretroviral therapies (ART) have transformed the once deadly HIV/AIDS disease into a manageable, chronic infection. Yet, there are still a number of pressing problems associated with current ARTs, including the necessity of daily administration of HIV-1 medications, suboptimal treatment adherence, and the emergence of drug-resistant viral phenotypes. Therefore, there is a need for developing long-acting antiretroviral agents targeting clinically unexploited viral proteins to mitigate the above problems. HIV-1 capsid protein is a novel, attractive target as its plays multiple essential roles during the virus life cycle. GS-6207 (Lenacapavir, Gilead Sciences) is a recently discovered, first-in-class, long-acting, and ultra- potent HIV-1 capsid inhibitor. Recently completed phase 1 clinical trials (NCT03739866) have suggested advancement of GS-6207 into phase 2/3 clinical trials (NCT04143594/NCT04150068) with a six-month dosing interval. Our research objective is to elucidate structural and mechanistic bases for a highly potent antiviral activity of GS-6207 and exploit the knowledge obtained to develop second-generation inhibitors. For this, we have synthesized and examined the antiviral activities of GS-6207. Consistent with the multifaceted role of capsid in HIV-1 biology, the inhibitor potently (EC50 of ~55 pM) impaired incoming virus and exhibited a second, slightly reduced (EC50 of ~314 pM) antiviral activity during virus egress. Mode-of- action studies of GS-6207 revealed that the inhibitor blocks post-entry steps of infection by stabilizing and thereby preventing functional disassembly of the capsid shell in the cytoplasm of infected cells. In addition, GS-6207 interfered with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. Our x-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments have revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that strikingly stabilize the curved capsid lattice. Furthermore, our high-resolution x-ray structure of GS-6207 bound to a capsid hexamer enabled us to map drug-resistant variants in close proximity to the GS-6207 binding site. This information will be critical for rational design of second-generation inhibitors. We propose to extend these studies to better understand the multimodal, exceptionally potent antiviral activity of GS- 6207 during both early and late steps of HIV-1 replication. For this, we will pursue the following three specific aims: Aim 1 will elucidate structural and mechanistic bases for inhibition of post-entry steps of HIV- 1 infection by GS-6207; Aim 2 will dissect underlying mechanisms of inhibition of virus production and maturation by GS-6207; and Aim 3 will investigate the structural basis for viral drug-resistance to GS-6207 and rationally develop second-generation inhibitors with an enhanced barrier to resistance. Taken together, the proposed studies will dissect the multimodal antiviral mechanism of action of GS-6207, provide new insights into the viral biology of capsid, identify key inhibitor-capsid interactions and facilitate optimization of this class of compounds for their clinical use.

NIH Research Projects · FY 2024 · 2020-09

Project Summary The goal of this proposal is to identify the mechanism(s) by which PD-L1 reverse signaling in dendritic cells (DC) initiates DC trafficking, T cell priming and T cell programming during cutaneous infection. Our studies have outlined a major role for PD-L1 reverse signaling in the control of DC migration from the skin to the draining lymph node. Intriguingly, this loss of migration appears to be dependent on TLR stimulation or infections that initiate type 1 IFN signaling. These findings are consistent with PD-L1 acting to mitigate type 1 IFN signaling events. We also outline a requirement for PD-L1 reverse signaling in chemokine, but not S1P, responsiveness demonstrating a new role for PD-L1 in regulating chemokine signaling. Finally, we find T cell priming in the lymph node is significantly decreased in the absence of PD-L1 reverse signaling. However, these defects in T cell priming only occur when DC trafficking is required, and not when antigens drain directly through the lymphatics and to the LN nor following systemic infection. Therefore, in this proposal we aim to better understand the requirements for PD-L1 reverse signaling in dendritic cell transmigration through the lymphatic capillaries. We also aim to understand how the extracellular and intracellular domains of PD-L1 may control responsiveness to chemokines. Finally, we aim to address the contribution of DC retention in the skin caused by loss of PD-L1 signaling to tissue resident memory responses established after vaccinia scarification.