University Of Colorado Denver

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that impacts approximately 1.5% of children in the United States. Individuals with ASD experience deficits in social communication or restricted interests and repetitive behavior; but the severity and patterns vary greatly and convey lifelong impairment for some. It is unclear how the presentation of ASD changes from early childhood into adolescence or adulthood. The causes of ASD are also unknown, though substantial evidence supports the contribution of both genes and environmental factors. These gaps in knowledge exist because US studies to date have lacked the sample size, depth of data collection, or appropriate life course timing to address these questions. The Study to Explore Early Development (SEED) is now able to address these prior limitations. SEED is a large case- control study of children ages 2-5 years and their families, implemented across eight states over three phases. SEED collected detailed data on children's core ASD symptoms, cognitive status, and presence of co- occurring conditions in early childhood, along with extensive risk factors related to maternal health and the perinatal environment as well as genomics. The SEED sample includes 2044 children with ASD, 1950 children with non-ASD developmental disabilities (DD), and 2285 population control children (POP), making this the largest etiologic study of ASD in the US. Recent ancillary studies - the SEED Teen Pilot and SEED COVID studies -- will soon add data on adolescent health and the consequences of the pandemic, respectively, for some SEED participants. The work proposed here, SEED Follow-up Studies (SEED FU), will maximize the impact of extant SEED data through analyses that characterize ASD phenotypes and assess the potential interplay between genetic and modifiable risk factors. SEED FU will also facilitate new data collection in middle childhood, adolescence and early adulthood to characterize changes in ASD phenotype across developmental stages, and the associated health, educational, and service needs across the early life course. These data will further enable prospective analyses of associations between early life factors and later childhood through early adulthood outcomes. Studying risk factors in relation to life course phenotypic subgroups may also help elucidate etiologies previously masked in ASD case-control studies. The NC SEED Team in combination with the SEED Network's collaborative infrastructure and extensive extant data resources, will ensure the successful implementation of the SEED FU Study in North Carolina and contribute to success across the network. SEED is well-powered for making significant contributions to our understanding of the complex autism phenotype and identifying factors associated with ASD risk in the population. The knowledge gained by SEED FU will greatly advance our ability prevent adverse developmental outcomes and to support individuals with ASD and their families to ensure optimal wellbeing through early adulthood.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT: Improving the timeliness and quality of care for lung cancer (LC) and head and neck cancer (HNC) patients is of utmost importance because delays in treatment initiation are associated with cancer recurrence, lower survival, and poor patient outcomes (e.g., distress). Rural LC and HNC patients experience significant treatment delays. Compared to urban patients, patients in rural areas face added challenges such as needing to travel long distances to access care, having lower socio-economic status, and having less availability of cancer treatment. The objective of this study is to improve the timeliness and quality of care for LC and HNC patients who are underserved (e.g., low-income, underinsured) and from rural and frontier counties in the Rocky Mountain States, treated by the University of Colorado Health System that serves patients from Colorado and Wyoming and the Sisters of Charity of Leavenworth Health System that serves rural patients from Colorado, Utah, and Montana. We will apply the CARES (Cancer Advocacy, Resources Education, and Support) intervention to target factors specific to rural LC and HNC patients who are underserved and factors that due to their statistical and clinical significance are associated with suboptimal initiation of treatment and poor quality of care for these patients. The hallmark of the CARES intervention is implementing it at key points during LC and HNC treatment, likely augmenting its effects on the outcomes more effectively and more efficiently than current usual care practices. Using a randomized controlled clinical trial (RCT) design, we will compare the CARES intervention effects to the effects of usual care practice on the: (a) time to treatment initiation and (b) time to treatment completion. The CARES intervention also targets (c) quality of care outcomes (e.g., patient communication, coordination of care, providing information to patients) and (d) patient-reported outcomes (e.g., coping, distress, quality of life). Approximately 440 LC and HNC patients will be recruited and randomly assigned to either the intervention (n=220) or to the usual care condition (n=220). We will employ intent-to-treat analyses with linear mixed models (LMMs) to analyze the primary outcome of time to treatment initiation and the secondary outcomes. We predict that those who receive the CARES intervention will improve in all study outcomes to a greater degree than those who received usual care. Our ultimate goal is the dissemination and implementation of the CARES intervention into rural clinical practice to improve the timeliness and quality of care for rural and underserved patients and reduce disparities in LC and HNC morbidity and mortality. Thus, we will assess the pragmatic implementation and scalability of the CARES intervention by evaluating the overall effectiveness of the intervention's strategies, as well as "how and why" they work in real-world practice.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) is the leading cause of death in kidney transplant recipients (KTRs) and death from CVD is the leading cause of graft loss. KTRs demonstrate abnormal endothelium-dependent dilation (EDD) and large artery stiffness, key pathophysiological antecedents to the development of CVD. Acid retention is a common feature of patients who have received a kidney transplant. KTRs have a single kidney and a decreased number of nephrons leading to an inability to excrete the daily dietary acid load. Additionally, KTRs receive several medications that can result in acid retention including calcineurin inhibitors. Acid retention results in increased ammoniagenesis leading to activation of the alternative complement pathway. Activation of the alternative complement pathway increases inflammatory factors, collagen deposition and endothelial inflammation contributing to tubulointerstitial damage and vascular dysfunction. We show that complement activation fragments are increased in KTRs compared to healthy controls and are inversely correlated with eGFR and EDD. Lower serum bicarbonate levels, even within the normal laboratory range, in KTRs are associated with an increased risk of graft loss, cardiovascular events and mortality. Small interventional trials have shown that treatment with alkali therapy slows progression of kidney disease, even in patients with normal serum bicarbonate levels. In our preliminary data, alkali therapy improved vascular endothelial function in 20 patients with chronic kidney disease stage 3-4. Because acid retention is common in KTRs, it is plausible that alkali therapy in KTRs may also result in improved vascular and graft function. In our preliminary data, we show in a randomized, double-blind, placebo-controlled crossover safety and feasibility study that sodium bicarbonate therapy is safe and feasible in KTRs and there is a trend towards improved EDD. We are proposing a randomized, double-blinded, placebo-controlled, 12 month study in 120 KTRs to examine the effect of sodium bicarbonate therapy on surrogate markers of CVD and graft function. Our overall hypothesis is that treatment with bicarbonate will improve indicators of vascular and graft function in KTRs by decreasing complement activation. In Aim 1, we will compare changes over time in brachial artery flow- mediated dilation and arterial stiffness, measured by aortic pulse wave velocity, before and after 12 months of sodium bicarbonate therapy or placebo. In Aim 2, we will compare changes over time in tubular atrophy and interstitial fibrosis in kidney biopsies before and after 12 months of sodium bicarbonate therapy or placebo. In Aim 3, we will examine changes in plasma and urine complement activation fragments (Ba and sC5b-9) and complement deposition in kidney tissue before and after 12 months of sodium bicarbonate therapy or placebo. The results of this novel study have the potential to inform clinical practice by providing the necessary evidence to establish sodium bicarbonate therapy as an inexpensive and easy to administer option for the treatment of vascular dysfunction and graft function in KTRs.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Mitochondria function is tightly regulated by a complex, intertwined machinery that dictates mitochondrial gross morphology, transport of organelles, inner membrane and cristae morphology, communication between adjacent mitochondria and contact with other organelles. These processes are collectively known as mitochondrial dynamics. My overall goal is to reach a comprehensive understanding on the mechanisms that govern mitochondrial dynamics and the interplay between mitochondrial transport with other aspects of mitochondrial and cellular biology. While the mechanisms of mitochondrial transport had been largely characterized in neurons, our recent advances showed that transport of energetically active mitochondria to the cortical cytoskeleton supports lamellipodia dynamics, fuels turnover of focal adhesion complexes and increases velocity and distance of random cell migration in epithelial cells. Because specialized cell types have unique spatiotemporal needs for mitochondria functions, a fundamental question is how similar are the mechanisms of mitochondrial movement between neurons and other cell types? Here we will focus on characterizing the function and regulation of alternative isoforms of trafficking proteins that are expressed in non-neuronal cells. Adding complexity to this picture, we have evidence of multifunctionality amongst trafficking proteins in non-neuronal cells. These novel functions include mitochondrial metabolism and mitochondrial signaling. This poses an important question: how are these multifunctional proteins regulated so single or double functions are enabled at a particular time and location? Overall, my research program will lead to discoveries on the regulation of mitochondrial trafficking and mitochondrial signaling pathways in non-neuronal cells. Because mitochondria are key organelles that maintain cellular homeostasis, and mitochondrial dysfunction are associated with neurological and metabolic diseases, cancer and aging, our advances have the potential to inform how to exploit these mechanisms for practical applications in medicine.

NIH Research Projects · FY 2025 · 2021-06

One of the remaining fundamental challenges we face in pharmacology is deciphering the mechanisms of action of general anesthetics (GAs). A complete anesthetic state involves loss of consciousness (hypnosis) and movement (immobilization), as well as loss of pain sensation (analgesia) and recollection of the event (amnesia). It is believed that GAs act through the multiple but specific proteins on neuronal membrane and different ligand-gated and voltage-gated ion channels have received a significant consideration. One of the compelling reasons to study voltage-gated calcium channels (VGCCs) in the mechanisms of anesthetic actions is that these channels are essential in regulation of synaptic transmission and excitability in the neuronal sleep pathway (e.g. thalamus) and in the brain regions involved in learning/memory (e.g. hippo- campal formation). Importantly, our previous studies have established that low-voltage-activated subtype of VGCCs or T-type calcium channels (T-channels) are inhibited by different classes of GAs within the clinically relevant concentration range. For the past two decades our work has established the role of the family of T-type VGCCs in acute and chronic pain processing, including post-surgical pain. However the role of VGCCs in the mechanisms of GA-induced hypnosis and amnesia remains elusive. Furthermore, despite substantial progress that has been made in the last two decades towards our understanding of how GAs act at the molecular level, much less is known about how GAs cause hypnosis and memory deficit at the level of intact neuronal networks. Hence, this proposal aims to elucidate the contribution of specific subtypes of T-channels to anesthetic effects in the thalamocortical (Research area 1) and hippo- campal circuitry (Research area 2). We will take advantage of mouse genetics, selective knock-down of different T-channel isoforms ex vivo and in vivo electrophysiology, optical recordings, as well as a battery of behavioral tests to address these key challenges. Our proposed work has the potential to overturn ex- isting dogma about anesthetic mechanisms and to shift the focus to underappreciated targets, such as neuronal T-channels. We posit that understanding the neurophysiological mechanisms of action of GAs that target T-channels may be used as a starting point to develop novel and potentially safer approaches and practices in clinical anesthesia. MIRA mechanism is well suited to achieve our stated goals because of flexibility to pursue new avenues within the research area of interest to NIGMS. Consistent productivity of our lab and our ability to collaborate with others in the field of anesthetic pharmacology strongly suggest that our approach will be fruitful. The proposed work is innovative in that new mechanisms of useful clinical effects of general anesthetics such as loss of consciousness and amnesia will be characterized. It is med- ically significant because it describes the importance of drugs that target voltage-gated calcium channels for potential development of safer practices in clinical anesthesia.

NIH Research Projects · FY 2026 · 2021-06

PROJECT SUMMARY/ABSTRACT Overview. Centrioles and their associated centrosomes are crucial cellular organizers that maintain tissue health and support development. They control the cell's microtubule (MT) network, which provides the framework for multiple cellular functions: intracellular trafficking, signaling, genome segregation, cell morphology, and cell mobility. Throughout the cell cycle, centrioles change through new assembly and modifications to their scaffolding functions. In G0/G1, centrioles nucleate a primary cilium for cell sensation and signaling. During S- phase, they duplicate once and only once alongside the genome, and in mitosis, they organize the mitotic spindle. These mechanisms become disrupted in both cancer and trisomy 21 (T21, which causes Down syndrome). Further, in specialized multiciliated cells, centrioles are amplified and have an additional role in nucleating motile cilia that generate fluid flows essential for reproduction, development, and respiratory function. Despite their importance, many aspects of centriole control, function, and mechanical force resistance remain poorly understood. Goals for five years. Our research program comprises three main projects investigating centriole assembly, function, and regulation. RNA processing and RNA-binding proteins have emerged as critically important components of the centriole regulatory machinery. Project 1 will investigate the relationship between centrosome-associated RNAs, RNA- binding proteins, centrosome translational regulation, cell cycle control, and centriole duplication. We will study two RNA-binding proteins: UNK, which controls local translation during PLK4-induced centriole duplication and Big1, which modulates the Tetrahymena cell cycle and centriole number control. Project 2 will examine how trisomy 21 in Down syndrome affects cilia formation, specifically focusing on T21 repression of CP110 uncapping and the relationship between cilia-dependent Sonic Hedgehog and PKA signaling. Project 3 explores centrioles as force capacitors for motile cilia, examining both their resistance to mechanical forces and their role in promoting efficient ciliary movement and hydrodynamic flow. How triplet MT inner junction proteins and MT post- translational modifications strengthen centrioles and modify the ciliary wave form will be investigated. Vision for the program. Our research into centriole and centrosome biology, particularly regarding MT-dependent trafficking and mechanical force resistance, will advance our understanding of mechanobiology, development, and disease. RNA metabolism plays a vital role in development, and identifying mRNA processing events affecting centrioles and the MT network will help us understand how different cell types utilize their MT networks. Additionally, our T21 studies reveal promising directions for understanding cellular mechanisms in cardiac, immune, and secretory cell systems affected by Down syndrome.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY The human gammaherpesviruses (γHVs) Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus are DNA tumor viruses that establish a lifelong infection. These viruses are strongly associated with pathogenic outcomes in immune suppression, including the AIDS defining malignancies Kaposi's sarcoma and non- Hodgkin lymphoma. A major challenge of the γHVs is that these viruses present a lifelong risk for viral re- emergence and pathogenesis, especially in the context of acquired or medically-induced immunosuppression. Lifelong infection is further confounded by the limited therapeutic interventions against γHV diseases. To date, the best defense against γHV-induced disease is an intact immune system. Despite this correlation, there remain major knowledge gaps in our understanding of: 1) protective aspects of successful vaccination to γHV infection, and 2) consequence of vaccination on the composition and frequency of infected cells, either before or after established infection. In this R01 application, we seek to investigate how vaccination alters γHV infection, using murine gammaherpesvirus 68 (γHV68), a mouse model of γHV infection, that facilitates study of infection and associated responses from primary infection through lifelong latency and re-emergence of lytic infection. We recently undertook studies to characterize how latency is regulated by viral and host factors at the single-cell level, identifying that the latent pool is heterogeneous based on expression of the latency- associated nuclear antigen (LANA), and that the proportion of LANA+ latently-infected cells is subject to regulation. These studies further identified that CD8 T cells, and the cytokine interferon-gamma, limit LANA+ latently-infected B cells, and that vaccination with a live-attenuated vaccine is capable of profoundly restricting LANA+ latent B cell infection in an interferon-gamma-independent manner. We now seek to investigate the role of myeloid cells in infection and vaccination on γHV infection both in vitro and in vivo. We will make use of vaccination protocols with defined differences in efficacy to identify the critical features using single cell analysis and high dimensional methods to distinguish the features of vaccine efficacy. We hypothesize that vaccination with a live-attenuated vaccine induces: 1) local and systemic myeloid reprogramming, which 2) redirects γHV myeloid infection into an immune-susceptible myeloid target. We will test this in three aims. First, we will analyze the impact of vaccination on local and systemic myeloid cells. Second, define how vaccination affects primary γHV infection of myeloid cells. Third, dissect how viral tropism is influenced by vaccine-induced effector mechanisms. By analyzing specific viral and host processes in the context of vaccines with varying efficacy, this proposal seeks to investigate myeloid reprogramming as a necessary, and therapeutically viable, target for vaccination against γHV latency.

NIH Research Projects · FY 2026 · 2021-05

PROJECT SUMMARY The long-term goal of this research program is to understand how specific axons of the developing central nervous system (CNS) are ensheathed with specific amounts of myelin, a specialized, proteolipid-rich membrane produced by oligodendroglia. During development, subpopulations of neural progenitors produce oligodendrocyte precursor cells (OPCs), which migrate and divide to populate the CNS. Subsequently, some OPCs differentiate as myelinating oligodendrocytes whereas other OPCs persist through adulthood. Importantly, myelin is plastic and can be modified by brain activity. Recent evidence indicates that changes in OPC proliferation, oligodendrocyte differentiation, myelin sheath characteristics and axon selection for myelination contribute to myelin plasticity. However, the molecular mechanisms that regulate myelination, particularly in response to neuronal activity, are poorly understood. The investigations that comprise this research program focus on three broad areas of developmental myelination. First, using single cell RNA-seq data we generated, we will investigate how neural progenitors are specified as OPCs. Second, will axo-glial interactions and mRNA localization promote myelin sheath growth. Third, we will investigate how microglia, the resident innate immune cells of the CNS, modify myelin coverage on axons in response to neuronal activity. We use zebrafish as a model system, which enables us to combine time-lapse imaging with genetic and pharmacological manipulations to observe and test cell behaviors and myelination in an intact, living animal. The results of this research program have the potential to provide important new insights to the developmental basis of learning, memory and psychiatric disease and to provide a foundation for designing therapeutic strategies to promote myelination of brains damaged by disease or injury.

NIH Research Projects · FY 2026 · 2021-05

Project Summary/Abstract Despite more than 40 RCT interventional trials in pregnant women at risk for delivering large-for-gestational age (LGA) infants, there is currently no clearly effective treatment to reduce fetal overgrowth in overweight/obesity (OW/OB), which account for ~70% of pregnancies. Maternal obesity remains the most common cause of LGA infants and increased fat mass at birth, the latter a stronger harbinger for the development of childhood metabolic disease. One in five preschoolers is already obese, and 40% already exhibit non-alcoholic fatty liver disease (NAFLD), suggesting early life adipogenic influences. We have shown that newborns from mothers with obesity and gestational diabetes are born with 68% more liver fat than those from normal-weight (NW) mothers, and earlier maternal TG, before subcutaneous fat stores have developed, predicted newborn liver fat. Non-human primate data support that liver fat at birth predicts later NAFLD. Our data show that under controlled conditions, OB mothers have 30-40% higher fasting and postprandial triglycerides (FTG, PPTG) throughout pregnancy. Moreover, FTG and PPTG are more predictive of newborn fat than glucose, BMI or fat mass, insulin resistance, or weight gain. Although maternal PPTG independently predicted 50% of the variance in newborn fat early (14 wks), by later pregnancy (28 wks) this effect was augmented by glucose. This suggests that rising glucose later in pregnancy stimulates fetal insulin (cord C-peptide), and when combined with excess TG availability, augments newborn fat storage. Although some data support TG in fetal overgrowth, TG are not measured as part of routine obstetric practice. In part, this is due to prior unavailability of a portable TG meter (similar to a glucometer) that allows repeated testing, which we have now successfully piloted. In this prospective cohort trial in OW/OB pregnancies we will, for the first time, obtain repeated measures of TG and glucose (by CGM) to define: 1) at what level of TG the risk of fetal overgrowth increases, and if this occurs independent of or in synergy with glucose; 2) when in pregnancy the TG exposure is most important, 3) if fasting vs postprandial TG results in greater newborn subcutaneous fat (Specific Aim 1; by air-displacement plethysmography) or in newborn liver fat (Specific Aim 2; by magnetic resonance spectroscopy), independent of other risk factors and accounting for sex differences. In our Exploratory Aim, we will interrogate mechanisms by which placental lipid transport pathways may facilitate fetal fatty acid (FA) delivery, the lipidomic signatures of maternal and cord blood which correspond with increased fetal fat accretion, and the adipogenic potential of umbilical mesenchymal stem cells. Completion of this community-based trial may provide compelling evidence to support a paradigm shift in obstetric practice that endorses meter TG monitoring, similar to glucometers in gestational diabetes, for mothers at risk for fetal overgrowth. Clinically impactful, these data may inform a future interventional trial in which TG are targeted with safe TG-lowering agents (i.e., high dose omega 3-FA supplements) to prevent excess newborn subcutaneous and liver fat, with the goal of decreasing childhood risk for obesity, NAFLD, and metabolic disease.

NIH Research Projects · FY 2025 · 2021-05

Abstract The low to moderate success with childhood obesity interventions to date and the persistent obesity disparities across race/ethnicity and socioeconomic status indicate the need to approach childhood obesity in a new and innovative way. Building on the last three decades of research on childhood obesity, the main objective of the proposed study is to utilize state-of-the-art intervention methods including ecological momentary intervention (EMI), video feedback, and home visiting methods in partnership with primary care clinics and Community Health Workers (CHWs) to examine whether increasing the quality of family meals (i.e., dietary quality, interpersonal atmosphere) and quantity of family meals (i.e., frequency of meals) reduces childhood obesity. Numerous studies have shown significant associations between family meal frequency and child weight and weight-related behaviors (e.g., better diet quality, lower weight status). Research has also shown that the quality of family meals, including dietary quality of the food served at family meals and the interpersonal atmosphere during family meals, is associated with decreased childhood obesity risk. In addition, prior intervention research has shown that immediate feedback on health behaviors (e.g., EMI, video feedback) increases the likelihood of behavior change. Thus, the proposed individual randomized controlled effectiveness trial, based on our pilot study, tests combinations of the above factors (i.e., EMI, home visiting, video feedback) across three study arms: (1) EMI; (2) EMI+Home Visiting (HV); and (3) EMI+HV+Video Feedback. All arms will receive 16 weeks of EMI family meal tip messages delivered via smartphones. Arms 2 and 3 will additionally receive home visiting (eight in-home visits; eight “Try it Yourself” activities) focused on family meal quality and quantity and a family meal prep activity delivered by a CHW simultaneously with the 16 weeks of EMI. Arm 3 will additionally receive eight weeks of video feedback focused on family meal behavior(s)/patterns delivered by a CHW during the eight in-home visits. All arms will receive an 8-week maintenance phase allowing for progressively less support of families so they can increase self-efficacy and sustainability of behavior change. The intervention will be carried out for 6 months with children with overweight/obesity (i.e., BMI ≥85%ile) who are ages 5-8 years (n=510), from low income and diverse households (i.e., African American, Hispanic, Native American, White), and their families. Eligible children will be recruited through primary care clinics. Drawing on Family Systems Theory, the intervention aims to change individual and family-level behaviors. Specifically, the intervention will be delivered to the family unit and primary outcomes will include child weight (i.e., BMI %ile) and diet quality (i.e., Healthy Eating Index). Secondary outcomes will include parent and other family member’s weight and weight-related behaviors. This study will change clinical practice by creating a new model for childhood obesity treatment in primary care using CHWs as interventionists and mobile health technology to intervene in real-time on parental stress and the home food environment to reduce childhood obesity.

NIH Research Projects · FY 2025 · 2021-05

Project Summary The short lifespan of human somatic cells, including adult stem cells, in ex vivo settings is a significant hurdle for many clinical and research applications. Therefore, the development of novel approaches that can facilitate the expansion of adult cells and at the same time restore the young-like characteristics of these cells without permanent immortalization is of high priority for regenerative medicine. Telomere attrition is one of the well-characterized mechanisms responsible for the decline in proliferation and function of somatic cells in culture and can be counteracted by the activity of the enzyme telomerase. Mitochondrial dysfunctions and epigenetic changes are also among mechanisms responsible for cellular aging, senescence and decline in functionality. In our preliminary experiments, we developed a patent-pending non-integrating RNA-based cocktail of factors that upon transfection into human somatic cells increases the length of telomeres to that observed in pluripotent stem cells, improves the proliferation rate of cells, restores mitochondrial DNA content and allows for a clonal expansion of individually seeded adult human fibroblasts (FBs). We now propose to use this rejuvenating RNA cocktail to develop two novel cellular technology platforms. One will improve the expansion of human primary somatic cells and promote their rejuvenation in a clinically relevant manner without permanent immortalization, while maintaining the proliferative capacity, functionality and normal characteristics of these primary cells. The second platform will allow for the efficient clinically relevant genetic engineering directly in primary somatic cells. In the latter, the use of our rejuvenating RNA cocktail will allow for the clonal expansion of genetically modified primary somatic cells, while preserving the functionality of these cells for subsequent transplantation. To develop the cell expansion platform, in Specific Aims 1 and 2, we will further characterize the effect of our cocktail on three human cell types commonly used in research and clinical settings: FBs, keratinocytes (KCs) and mesenchymal stromal/stem cells (MSCs). We will perform detailed molecular and functional characterizations of low and high passage human FBs, KCs and MSCs treated with our cocktail with a focus on the restoration of young-like characteristics of these cells. In Specific Aim 3, we will employ our rejuvenating cocktail to develop a platform for the generation of genetically modified human somatic cell lines. Using Cas9-mediated gene targeting, we will generate a panel of primary human FB, KC and MSC lines with the site-specific knock-in of fluorescence reporters and luciferase. The derived lines will be tested ex vivo and in vivo to confirm their functionality and safety, providing feasibility data for the development of novel somatic cell gene therapies for many diseases. If successful, the studies will serve as a proof of principle for developing rejuvenating strategies and genetic engineering platforms for other somatic cell types used for transplantation, such as hematopoietic and muscle stem cells.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Twenty-five percent of children with congenital heart disease (CHD) undergo intervention in the first year of life, often requiring surgery with cardiopulmonary bypass (CPB). CPB and related techniques including deep hypothermic circulatory arrest (DHCA) are necessary but contribute to poor postoperative physiology. Mortality for high risk surgeries remains >10%. Major complications occur in 30% of these complex surgeries and are key drivers of hospital length of stay (LOS) and costs. Novel diagnostic, mechanistic, and therapeutic approaches are critically needed to impact this burden on our infants, families, and healthcare system. Metabolites are the small-molecule end products of the genome that collectively determine minute-to-minute cellular physiology. Individual metabolites (e.g. lactate) are commonly used in postoperative management, but the interrelated metabolomic changes induced by infant cardiac surgery remain poorly understood. Recently, the metabolic profile of infants undergoing CPB was shown to shift markedly during the first 24hrs postoperatively and metabolites from two related pathways (kynurenine and nicotinamide metabolism) were associated with mortality and ICU LOS. Evolution of the postoperative metabolic profile beyond 24hrs and comprehensive changes in circulating/tissue kynurenine and nicotinamide metabolites are unknown. Overall Hypothesis: Infant cardiac surgery with CPB induces pathologic changes in the circulating metabolome across multiple key metabolic pathways. These changes directly impact postoperative outcomes and organ injury through a combination of beneficial metabolite depletion and pathologic metabolite production. Proposal: The study will use a combined clinical and translational approach. The clinical arm will consist of a prospective cohort study of infants undergoing CPB, with serial targeted metabolic profiling and pathway mapping through 72hrs postoperatively. The complementary translational arm will consist of a piglet model of CPB/DHCA to evaluate and modulate organ-specific flux through kynurenine and nicotinamide metabolism. Specific Aim 1: Validate the association of the 24hr postoperative metabolic profile with the combined outcome of death, cardiac arrest, or mechanical circulatory support and determine the evolution of this pathologic metabolic profile through 72hrs postoperatively. Specific Aim 2: Perform quantitative mapping of the kynurenine and nicotinamide metabolic pathways in order to a) quantify individual metabolite abnormalities, b) identify contributing changes in pathway enzymes, and c) determine the association of specific pathway abnormalities with postoperative outcomes. Specific Aim 3: In a piglet model of CPB with DHCA, quantify circulating and organ-specific kynurenine and nicotinamide pathway metabolites and determine the effects of pathway blockade on development of postoperative acute organ injury using systemic indoleamine 2,3-dioxygenase inhibition.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Epigenetic mechanisms play a pivotal role in aging and are found disregulated in age-related disorders, including neurodegenerative diseases and cancer. The major indicator of alterations occurred in chromatin during aging is acetylation of lysine 16 of histone H4 (H4K16ac), a modification that is redistributed and raised in the healthy aged brain but is considerably lost in Alzheimer’s disease. On the molecular level, H4K16ac is involved in a wide array of fundamental cellular processes, including higher-order chromatin decompaction and folding, DNA damage repair, and gene expression. Despite the high importance of H4K16ac, very little is known about protein ligands that bind this mark. Our recent studies identified the plant homeodomain finger 6 of the histone methyltransferase MLL4 (MLL4PHD6) as a selective effector (or reader) of H4K16ac. The molecular mechanism underlying the recognition of H4K16ac by MLL4 is unknown and will be elucidated in the proposed studies. We hypothesize that the selective targeting of H4K16ac by MLL4 at specific genomic sites is necessary for transcriptional activation of MLL4 target genes and that this interaction provides a novel functional link between MLL4 that methylates lysine 4 of histone H3 (H3K4) and the acetyltransferase MOF that produces H4K16ac. We seek to define the molecular basis and functional significance of the previously uncharacterized crosstalk between vital histone marks. These studies are fundamental to our understanding of physiological activities associated with ‘writing and reading’ H4K16ac and are also essential to better understand the etiology of human age-related illnesses, including AD and other neurodegenerative disorders.

NIH Research Projects · FY 2025 · 2021-04

Summary Clostridioides difficile infection (CDI) is an important cause of morbidity and mortality and rates are on the rise, indicating that safe and new approaches are urgently needed for treatment and prevention. Emerging evidence suggests that a high-fat/low-fiber diet may promote CDI. Diets high in saturated fat lead to the production of primary bile acids that can promote infection by germinating C. difficile spores. Diets deficient in fiber perpetuate C. difficile colonization in mice, and this effect was linked at least in part with a loss of Short Chain Fatty Acids (SCFAs). Our preliminary murine studies show that a high-fat/low-fiber diet resulted in increased microbiome disturbance following broad spectrum antibiotic challenge, increased cecal levels of primary bile acids that germinate C. difficile spores, markedly decreased levels of secondary bile acids that can kill C. difficile, and increased morbidity and mortality upon C. difficile exposure. These results suggest that dietary intervention has promise for preventing CDI in individuals at high risk. Aim 1A will determine the effects of dietary levels of fat and fiber in preventing antibiotic induced gut microbiome disturbance and CDI, using conventional mice fed varied diets. Aim 1B will directly evaluate the role of increased intestinal levels of primary bile acids in the increased C. difficile pathogenicity by chemically inhibiting the ileal apical sodium-dependent bile salt transporter. Oncology patients have high incidence of CDI, driven by risk factors that include frequent hospitalization, antibiotic use, and use of chemotherapeutic drugs. Aim 2 will test a higher-fiber/lower-fat dietary intervention for prevention of C. difficile recurrence and maintenance of gut microbiome diversity in oncology patients. Production of SCFAs may be one mechanism contributing to the protective effects of fiber in CDI. Metabolism of the SCFA butyrate by intestinal epithelial cells plays a key role in the establishment of intestinal hypoxia, which is important because reversion to hypoxia is a key process in promoting the reestablishment of an anaerobe dominated complex gut microbiome following disturbance. SCFA production from fiber is limited in individuals with a low complexity facultative anaerobe- dominated microbiome, which is common in individuals with recurrent CDI. In our earlier work, we have identified butyrate-producers, including Clostridium symbiosum and Anaerostipes caccae that specialize to infant and disturbed guts and that can produce butyrate using a simple substrate, gluconic acid, as a sole source of carbon. Thus, in Aim 3 we will test the hypothesis that synbiotic treatment with disturbance adapted butyrate-producers and gluconic acid will increase butyrate production, increase intestinal hypoxia and facilitate the activity of anaerobic secondary bile acid producers that prevent CDI, using mice humanized with a disturbed/ low-complexity microbiota.

NIH Research Projects · FY 2026 · 2021-04

Project Summary Asthma remains a prevalent public health concern that disproportionately affects children living in urban low- income settings. Previous studies have identified multiple pathobiological subtypes (i.e., endotypes) of childhood asthma, notably the airway Type 2 inflammation high (T2-high) asthma endotype. Endotypic understanding of asthma has led to better asthma management through personalized therapy. Yet, asthma endotype research is still in its infancy, and the pathobiologic mechanisms driving disease in many children remains to be determined. In particular, the timing and mechanisms of airway endotype development and relationships to lung dysfunction are unexplored. The University of Colorado School of Medicine CAUSE Clinical Research Center project WINDOWS seeks to identify critical windows in airway endotype development that lead to lung dysfunction and disease, providing potential targets for asthma prevention. WINDOWS is an early-life longitudinal cohort study of high-risk urban children that maps the molecular steps in the development of airway endotypes, which lead to early-life lung function deficits, and eventually persistent childhood asthma. We are recruiting toddlers age 1.5-3 years from urban, low-income families with a history of at least three wheezing or asthma episodes requiring treatment to enrich for children at highest risk of asthma persistence. They will be followed over four years with annual airway molecular endotyping and lung function assessments to age 6-7 years, when persistent asthma status will be ascertained. Sequential RNA sequencing of nasal airway samples obtained during early childhood will reveal new insights into the mechanisms underlying both T2-high and non-T2-high asthma, which will be coupled with measures of airway obstruction using the forced oscillation technique and impedance pneumography. Comparing subjects that do and do not develop asthma will allow us to distinguish airway endotype and lung function phenotype features associated with progression from an early life high risk state to childhood asthma. The aims of WINDOWS are to 1) characterize trajectories of lung function and progression of wheezing phenotypes from early life through childhood in a cohort of toddlers at high risk for developing childhood asthma; 2) transcriptionally characterize the airway epithelium from early life through childhood in toddlers at high risk for developing childhood asthma; and 3) determine the relationship between airway transcriptome profiles and measures of lung function in early life and how these early life molecular and physiologic features relate to the clinical characteristic of childhood asthma. WINDOWS will provide insights into the role of airway dysfunction in the development of childhood asthma by using innovative methods to investigate airway biology and physiology in early life, which will fill the gap in understanding airway resilience and asthma prevention.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT The overall goal of our proposed research is to understand why having health care coverage (or eligibility for health care coverage) is not sufficient to allow equal access to elective surgical care. Despite successful efforts to expand coverage through Medicaid expansion and the Affordable Care Act (ACA), potentially elective surgical care is often not addressed until it becomes an emergency. These patients tend to come from vulnerable populations, who not only present more often for Emergency General Surgery (EGS), but experience worse outcomes and greater costs. The disparities leading to this presentation in the United States have been well-described in terms of overall relationship to insurance status, race and income, but deeper data collection and analysis are desperately needed to identify modifiable factors that can inform interventions around decreasing emergent presentation in these populations, particularly in regard to health care coverage. Even in countries with Universal Health Care systems, disparities are noted in how people are able to actually access those services. We initially studied this problem in the context of emergent cholecystectomy, one of our most common presentations of EGS disease. We found that lack of health care coverage was not a major factor; in fact, 86% of our patients had some type of coverage (29% private, 57% public), and many other social factors led to an emergency operation. We now seek to expand and explore this in detail for the other EGS conditions defined by the American Association for the Surgery of Trauma (AAST) using a multiphase mixed method approach. We will 1) identify modifiable factors for emergent presentation and explore the trajectory of progression to elective versus emergency surgery using billing data and EHR in a convergent mixed-methods design, combining quantitative variables with qualitative narrative data, 2) identify and quantify additional modifiable factors from the patient perspective that are not available in clinical or administrative datasets using an exploratory sequential design, using identified domains to conduct systematic review and meta-analysis for quantitative data, and 3) determine which modifiable factor or factors identified will have the greatest impact for future intervention strategies using Markov modeling. This proposal will leverage our ability to link data from multiple sources in novel ways, our diverse, robust general surgery population in a Medicaid expansion state, and Co-Investigators who are expert in their fields of longitudinal data modeling and mixed methods research. With this data we can model and understand what influences the persistent disparity in the ability to access elective surgical care despite increased coverage, and predict which factors contribute the most to the disparities and thus hold potential for the greatest impact. By identifying actionable modifiable factors, we will ultimately inform effective intervention strategies to prevent emergent presentation of elective surgical disease.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY The long-term goal of this project is to study age-dependent mechanisms of follicle-stimulating hormone (FSH) actions in the ovary. FSH is a pituitary glycoprotein consisting of an α-and a β-subunit. Both the subunits are glycosylated with two N-linked sugar chains on each subunit. Glycosylation of FSH is estrous/menstrual cycle- and age-specific. Macro-heterogeneity results in FSH variants consisting of 2 sugar chains on the α subunit but either one or none on the β. These variants are known as hypo-glycosylated FSH glycoforms, FSH21, and FSH18, in contrast to the fully glycosylated FSH24. Interestingly, the abundance of hypo- and fully glycosylated FSH is age-dependent, with high levels of FSH21/18 glycoforms predominant in young women and FSH24 predominant in peri/post-menopausal women. This shift suggests a role of FSH24 in ovarian aging. In vitro studies and pharmacological rescue of Fshb null mice with recombinant hypo- and fully glycosylated FSH glycoforms indicate differences in regulation of known FSH-responsive ovarian genes and proteins downstream of FSH receptor (FSHR) signaling. However, the mechanisms by which these FSH glycoforms regulate ovarian signaling pathways in vivo are unknown. The central hypothesis is that age-specific FSH glycoforms act via FSHRs but regulate distinct downstream signaling cascades to elicit different gene/protein expression signatures in the ovary. This hypothesis will be tested using genetically engineered novel mouse models. In Aim 1, we will perform a trans-omics analysis by overlaying the gene, protein and phosphoprotein expression signatures in ovaries of Fshb null mice expressing individual FSH glycoforms to identify signaling networks regulated by each FSH glycoform. In Aim 2, we will test the hypothesis that the FSH glycoform specificity in FSHR-mediated signaling is achieved by recruitment of distinct protein complexes to activate different downstream gene/protein networks. Fshb null mice expressing individual FSH glycoform and His - tagged FSHRs in granulosa cells will be used. Pull-down experiments with an His tag-specific antibody followed by mass spectrometry analysis of ovarian proteins will allow us to identify the FSH glycoform-specific FSH receptor and receptor co-factor protein complexes in each case. In Aim 3, we will evaluate the direct effects of recombinant FSH glycoforms in secondary follicles obtained from reproductively young and old mice. Gene and protein expression profiling will be performed and how FSH glycoforms impact follicle growth and gamete quality in vitro will be determined. Successful completion of the proposed studies will advance our understanding of the mechanisms by which FSH glycoforms regulate selective recruitment of distinct FSHR - co-factor partner complexes to achieve FSHR-mediated signal transduction pathways in vivo in ovaries and provide a direct read out of FSH glycoform actions during in vitro folliculogenesis and oogenesis. Our mechanistic studies serve as the foundation for novel therapeutic opportunities to preserve ovarian function and will enable improved design of ovarian induction protocols, in alignment with the NICHD mission.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT Weight loss is associated with a reduction in obesity-related health risks, but can be difficult, with preventing subsequent weight regain even more challenging. As such, understanding mechanisms underlying energy balance regulation and identifying strategies for successful weight loss and maintenance are important goals, and are key components of the Strategic Plan for NIH Obesity Research. Food intake is a complex process involving homeostatic signals (e.g., appetite-related hormones) and non-homeostatic signals (e.g., reinforcing properties of food). One factor that may contribute to susceptibility to obesity is a high responsivity to high-calorie foods, which promotes increased caloric intake. Food preferences involve learned associations thought to develop via classical conditioning through repeated pairings with external stimuli. Improving our understanding of the neuronal mechanisms underlying these processes and attempting to modify them may be a useful strategy for weight loss and maintenance. Therefore, the proposed study aims to investigate the neuronal and behavioral effects of an intervention designed to alter affective associations with food, using a novel implicit priming (IP) paradigm, in which positively or negatively valenced images are presented immediately prior to food images, but not consciously perceived. We hypothesize that IP will alter neuronal and behavioral responses related to food intake, reducing the appeal of high-calorie foods and promoting weight loss and maintenance. The project goals are to further delineate the neuronal mechanisms underlying the intervention, establish the impact of IP on longer-term food preferences and eating behavior, and determine if it can facilitate weight- loss maintenance in individuals with overweight/obesity. Effects of IP on neuronal responses to visual food cues and measures of eating behaviors (food intake, preferences) will be measured not only acutely, but also following 12 weeks of weekly IP administrations, within the context of weight-loss maintenance. Weight and body composition will be measured before and after the intervention, and, to assess lasting effects, 12 weeks after the intervention has ended. Participants will be randomized to active IP, control IP (with scrambled images as primes), or to an active control, cue exposure therapy (CET). Sex-based differences will also be examined, as studies have observed women to have stronger, more frequent food cravings, greater neuronal response to hedonic food cues, and greater sensitivity to disgust than men. The use of neuroimaging in this study will provide a more sensitive measure than behavioral measures alone and will help to identify mechanisms through which the intervention changes behavior. If the project aims are achieved, it would not only yield new information about the neurobiology of food intake behavior, but also could represent a potential novel intervention for treatment and prevention of obesity.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Although mechanical overloading of joints has been implicated in the comorbid association between obesity and osteoarthritis (OA) [1, 2], we and others have established a pathogenic role for obesity-associated inflammation [3-6]. Our work to further study inflammation in this context has led to new data implicating dysbiosis of the gut microbiome as a root cause of inflammation in the colon, circulation, and synovium that culminates in accelerated OA degeneration in joints [7]. Changes include colonic, serum, and synovial upregulation of inflammatory cytokines, which parallel the expansion of Peptococcaceae and Peptostreptococcaceae family members in the obese gut. Correction of this dysbiosis via dietary supplementation with the indigestible prebiotic fiber oligofructose ablates these proinflammatory communities while restoring an Actinobacteria taxa that is lost in obesity, Bifidobacterium pseudolongum (B. pseudolongum). This correction leads to reduced inflammation in niches spanning from the colon to the joint, reduced numbers of macrophages and B cells in the synovium, and protection against the development of OA in the knee [7]. Moreover, we have discovered that oral delivery of a B. pseudolongum probiotic is joint protective and the B. pseudolongum metabolome itself contains molecules that directly inhibit inflammation. Based on these findings, we propose that 1) the OA of obesity is caused by a gut microbiome dysbiosis that triggers an inflammatory cascade starting in the intestine and radiating to the joint, and 2) obesity-related OA can be mitigated either by correcting the obese gut dysbiosis using methods to expand B. pseudolongum or by commandeering its metabolites to reduce inflammation in the colonic epithelium where the obesity-related inflammatory signature initiates. To investigate these concepts, we propose to address the following two Specific Aims. Aim 1 is to establish that gut microbiome dysbiosis is causal in the OA of obesity, with the hypothesis that the obese dysbiotic gut microbiome is the initiator of a systemic inflammatory cascade that initiates in the colon, radiates to joints, and accelerates OA. Aim 2 is to study how B. pseudolongum protects against joint degeneration in obesity, with experiments designed to test the hypothesis that B. pseudolongum mitigates inflammation and is joint protective in the context of obesity and its metabolome contains inflammation-suppressing agents. Completion of these aims will establish that the OA of obesity is an inflammatory process driven by gut microbiome dysbiosis. Expansion of B. pseudolongum or delivery of its metabolites could represent novel therapeutic approaches to address a disease of global scope that is currently only treated palliatively.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract. This application for a K01 award describes the research & mentoring plans and coursework for Dr. Alexander Kaizer, an Assistant Professor of Biostatistics and Informatics at the University of Colorado-Anschutz Medical Campus (CU-AMC), to achieve advancement towards independent research in the use of adaptive designs for cardiovascular outcome clinical trials that facilitate information sharing across different sources of data to improve the statistical efficiency of evaluating new therapies. The process to develop effective novel treatments traditionally proceeds through a series of studies and phases. Conventionally, each phase is treated independently from previous phases, which are traditionally only used in the design stage of a new trial. This represents a potentially inefficient use of all available data that could be incorporated beyond the design stage and represents an important limitation for newer trial designs that may include multiple treatments within the context of a single protocol, but where comparisons only use concurrently collected data. The statistical methodologies and trial designs proposed in this application address this limitation by developing new methods to facilitate information sharing along with applications to platform trial designs. In this award, the development of statistical methods will be coupled with formal training in the biology of the cardiovascular system to assure that these new methods have seamless application in the design of cardiovascular outcomes research. To achieve the training goals and research aims laid out in this K01, a research team of three mentors has been assembled. Dr. John Kittelson, Professor of Biostatistics and Informatics at CU-AMC, is an expert in clinical trial design and has extensive experience with cardiovascular trial implementation and analysis. Dr. Gregory Schwartz, Professor of Medicine at CU-AMC and Chief of the Cardiology Section at the VA Medical Center, is a leader in proposing, implementing, and disseminating cardiovascular outcome clinical trials. Dr. Robert Eckel, Professor of Medicine at CU-AMC, past President of the American Heart Association, and President-elect of the American Diabetes Association, is an expert in lipid and lipoprotein metabolism and diabetes. In Aim 1, we will develop statistical methods for incorporating data from supplemental sources, such as past trials, into the analysis of a current study based on their exchangeability (i.e., equivalence) after adjusting for covariates (also known as information sharing). In Aim 2, we will develop adaptive platform trial designs that consider new treatment arms compared to a shared control arm. To improve the accessibility of the new methods, user-friendly software will be developed (Aim 3). The methods and designs from these aims will be evaluated via rigorous simulation study to understand their small sample properties under various scenarios. Methods will be illustrated through application to previously conducted cardiovascular trial data available from the NHLBI BioLINCC. Together, these methods for information sharing and adaptive trial designs will improve the efficiency of the research process and take fuller advantage of available information for statistical inference.

NIH Research Projects · FY 2026 · 2021-04

Project Summary The goal of this two-site proposal is to determine whether and by what means insulin resistance, in the form of impaired insulin regulation of microvascular perfusion, leads to decreased functional exercise capacity (FEC) in type 2 diabetes (T2D). Data from our two research teams suggest that the cardiac and skeletal muscle microvascular dysfunction present in people with T2D contributes to limitations in cardiac and skeletal muscle oxygenation and function associated with impaired function exercise capacity (a major predictor of CV and all- cause mortality). Insulin action is a potent predictor of the FEC impairment in T2D. The exact relationship between insulin action, cardiac and muscle dysfunction, cardiac and skeletal muscle perfusion and decreased FEC in T2D remains unclear. Here we propose to address this gap in knowledge by defining the roles of impaired insulin-mediated cardiac and skeletal muscle perfusion and exercise performance in people with T2D. Hypothesis: Decreased insulin-mediated muscle perfusion found in T2D contributes to the development of cardiac and skeletal muscle dysfunction and subsequent impairment in exercise capacity. We further hypothesize that exercise training attenuates insulin resistance and restores insulin-mediated perfusion to the skeletal and cardiac muscle, leading to improved exercise performance. Specific Aim 1: To test the hypothesis that impairment in insulin-mediated cardiac perfusion limits exercise performance through its effect on cardiac function in people with T2D. We will examine the relationship between insulin- mediated cardiac perfusion, other measures of cardiac function, and VO2 peak at rest and with exercise in subjects with and without T2D. Given that there is a sex disparity in diabetes outcomes and exercise impairment with a lack of mechanistic insights on sex differences, we will analyze all parameters for differences by sex on an exploratory basis in the three aims. Specific Aim 2: To test the hypothesis that impaired insulin-mediated skeletal muscle perfusion limits exercise performance through its effect on oxidative capacity in people with T2D. We will examine the relationship between insulin-mediated skeletal muscle perfusion; muscle oxygenation, skeletal muscle mitochondrial function and in vivo skeletal muscle oxidative flux; and VO2 peak in subjects with and without T2D. Specific Aim 3: To test the hypothesis that the improvement in FEC subsequent to exercise training operates via action on cardiac and muscle function in T2D. These experiments will test whether the improvements in VO2peak observed with exercise training correlate with improvements in insulin-mediated perfusion, cardiac and skeletal muscle function and the impact of T2D on these changes. Understanding the role of microvascular disease in the diabetes-mediated exercise impairment may offer novel targets for intervention to improve exercise capacity, functional status and longevity in people with diabetes. Together, our two groups will employ complementary theoretical backgrounds and research methods in a synergistic approach to address the innovative hypothesis posed to improve health in T2D.

NIH Research Projects · FY 2025 · 2021-04

SUMMARY. Salivary gland cancer (SGC) is an orphan disease for which no targeted therapies are approved. SGCs are divided into histotypes, the most common being adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). We have generated one of the largest reported SGC PDX banks including major histotypes like ACC and the first reported PDX models of MEC. Our SGC PDX bank includes several cases from the same patient, collected after subsequent relapses, which has allowed an exploration of the acquisition of oncogenic gene events. Similar to other reports, our SGC models had low mutation burden by whole exome sequencing, and RNA-seq analysis identified known (MYB-NFIB), novel (NFIB-MTFR2), and even dual gene fusions in ACC cases that gave rise to oncogenic fusion protein products. Other genetic events in SGC clustered in the PI3K and mTOR pathways, which impinge upon protein synthesis. Both MEC and ACC overexpress key transcription factors such as Myc or SOX2 that dictated tumorigenicity and growth in SGC. Overall, we propose that protein synthesis is an unexplored target in SGC. SVC112 is a small molecule that inhibits protein synthesis at the elongation step by inhibiting eEF2. In our seminal studies where SVC112 was first reported, protein synthesis and growth inhibition were associated; SVC112 had greater effect on cancer over non-cancer cells; SVC112 depleted SOX2, Myc, and Cyclin D1, and arrested growth in vivo. In both ACC and MEC cell lines we found that SVC112 inhibits both native Myb and the protein products of MYB fusions and key proteins like Myc. SVC112 inhibited proliferation and sphere formation in both ACC and MEC in vitro assays, and had notable single agent activity (including actual tumor shrinkage) in 2 ACC PDX, one with a MYB fusion and another one showing non- fusion mediated Myb upregulation. We propose to study the role of oncogenic fusions in SGC, the mechanism of action of SVC112 in SGC, and the efficacy of SVC112 in complex SGC models. First, we will catalogue and prioritize MYB fusions from large patient and PDX cohorts. Then, to understand key genetic events we will insert fusions in non-cancer cell lines, and deploy CRISPR and lentiviral vectors in fusion-and non-fusion-bearing SGC cell lines, respectively; we will also test SVC112 inhibition to add an additional layer of testing of the impact of protein modulation. The hypothesis that SVC112’s selective effect is due to selective depletion of key proteins will be tested by ribosome profiling (to identify mRNA targets) and proteomics analysis (to identify proteins targets). Lastly, we will test the in vivo efficacy of SVC112 using both native SGC PDX models bearing MYB and other fusions and wild-tpy, as well as in vivo models with the CRISPR-depleted MYB fusions strains. Overall our goal is to examine dependence on fusion events vs protein overexpression by other molecular mechanisms for SVC112 susceptibility. This project will propel the translation of SVC112, a new drug for SGC discovered in Colorado, by dissecting the basis for its effect and therapeutic window, and identifying its efficacy in advanced SGC models with a plan that enables identification of which subset of SGC patients may derive more benefit.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Approximately 8 million adults in the US suffer from balance impairment due to damage to the peripheral vestibular system, but effective treatments for balance dysfunction are lacking. Vestibular hair cells within vestibular canal and otolith organs convert motion into receptor potentials and sensory information is relayed to the brain by action potentials (APs) in vestibular afferent nerves. Afferents in central zones (CZ) of vestibular neuroepithelia exhibit different responses to vestibular stimuli than afferents in peripheral zones (PZ). The nature of the neural code conveying vestibular information in distinct afferent types is poorly understood. There are 3 types of vestibular afferents: calyx-only afferents innervate one or more type I hair cells, bouton dendrites innervate type II hair cells and dimorphic afferents contact both hair cell types. Our goal is to elucidate distinct AP firing mechanisms in afferents with calyx terminals to better understand vestibular coding. Calyx-only afferents are present solely in CZ and have irregular firing patterns, whereas dimorphic afferents exist in both CZ and PZ and have regular firing patterns. To achieve our goal we will refine novel preparations of vestibular cristae and utricles, developed by our laboratory, as tools to study calyx-bearing afferents in CZ and PZ of gerbil neuroepithelia. Electrophysiological, hair bundle stimulation, immunohistochemical and pharmacological approaches will allow characterization of ion channels in afferent fibers in developing and mature epithelia. In Aim 1 we will determine the contributions of K+ channels and hyperpolarization-activated cyclic nucleotide- gated channels to AP firing in CZ and PZ afferents. Aim 2 will test the hypotheses that Nav1.6 channels with transient and resurgent characterisitics contribute uniquely to AP firing in mature PZ dimorphs. In Aim 3 we will incorporate ion channel data from Aims 1 and 2 into a novel, custom-written three dimensional mathematical model of the calyx to provide insight into our zonally-driven experimental findings. To determine how channel localization directly impacts AP firing, identified channel types will be strategically placed on the inner and outer faces of the calyx terminal and associated axon and channel density varied. Our results will clarify how sensory information is conveyed and how zonal encoding is generated within segregated vestibular afferents. Our data will inform development of vestibular neurotherapeutics targeting specific groups of ion channels in afferent nerves. Existing vestibular prosthetic implants attempt to restore normal vestibular function by direct electrical stimulation of vestibular afferents. A clearer understanding of AP generation and propagation within vestibular afferent sub-types is needed to inform appropriate electrical stimulation parameters. Results from this work could provide important new information on vestibular afferent coding and inform development of pharmaceutical and electrical strategies to combat vestibular dysfunction.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Parkinson’s disease (PD) is a neurodegenerative disorder associated with Lewy Body pathology that can lead to progressive cognitive decline and Parkinson’s disease dementia (PDD), a form of Lewy Body Dementia. Cognitive dysfunction in PD is common, affecting up to 80% of persons with PD (PwP) over the disease course. PD-associated cognitive decline has devastating consequences, including quality of life impairment, increase in caregiver burden, loss of independence and productivity, and increased risk of institutionalization. This results in significant public health and societal burden. Pharmacologic treatments for cognitive dysfunction in PD are often ineffective and none prevent progression to PDD. Exercise has promise for improving cognition in PD, but the best exercise prescription for individual PwP is unknown. Slow wave sleep (SWS) is important for cognitive function due to its involvement in synaptic plasticity, cortical reorganization, and glymphatic function. The PI’s preliminary data show that SWS is important for cognitive performance in PwP. Further, the PI found that exercise increases SWS in PD and only PwP with increased SWS had improvement in executive function. This interindividual response heterogeneity provides an opportunity to tailor exercise prescriptions to individual PwP. The hypothesis is that exercise will improve cognitive function in PD and that changes in SWS will serve as a biomarker and mediator of rehabilitation-induced cognitive response. Our exploratory hypothesis is that efficiency of glymphatic function may underlie these effects. Using an innovative Sequential Multiple Assignment Randomized Trial (SMART) design, the PI will test these hypotheses in a randomized, controlled trial of 120 PwP to investigate 1) the effects of exercise rehabilitation versus delayed-exercise control on cognition in PwP (Aim 1); 2) determine if changes in SWS due to exercise mediate the exercise-induced changes in executive function in PwP (Aim 2); and 3) determine if glymphatic function predicts exercise-induced changes in cognition (Exploratory Aim). Study design: In the first phase of the trial, participants will be randomized to 12-weeks of progressive resistance training rehabilitation (PRT) or delayed-exercise control (1:1). Arm assignment in the 2nd 12-week phase of the trial will be determined by individual response in the first 12 weeks. Specifically, responders (those with increases in SWS) will continue PRT for the 2nd 12 weeks of the trial while non-responders (no sufficient increase in SWS) will transition to endurance training (ET) for the 2nd 12 weeks. After the 1st 12 weeks of the trial, participants in the delayed-exercise group will perform PRT for the 2nd 12 weeks of the trial. The study addresses priority areas of NCMRR by investigating 1) an objective marker (SWS) that may predict individual rehabilitation treatment response and 2) treatment for secondary conditions (cognitive impairment) associated with physical impairment (Parkinson’s disease).

NIH Research Projects · FY 2026 · 2021-04

Abstract Physician-scientists serve a critical role in helping bridge the gap between basic and translational research and as noted in PAR-20-094 “bring a unique perspective to research through [a] blend of clinical and research experiences.” Thus training Psychiatrists for future careers as physician-scientists is significant and aligns well with NIMH priorities to “strive for prevention and cures” and “increase the public health impact of research.” This proposal seeks to recruit 2 promising physician scientists per year, with careful attention to diversity, into the Pathways-RRT, a longitudinal research program within the University of Colorado Psychiatry residency. The Pathways-RRT allows residents to complete all ACGME requirements for on time graduation and to sit for board certification, while also creating individualized pathways for research skills development and productivity during residency. The program provides between 14 and 18 months of research-dedicated time across residency (amount of time depends on whether applicants enter Child Fellowship training where they will continue their research training). Residents will be recruited based on their qualifications, demonstrated passion for research, and in part, upon their areas of interest being in sync with our current and growing pool of outstanding primary research faculty mentors. Program participants receive structured didactics focusing on research design, biostatistics, rigor and reproducibility, the ethical conduct of research, and grant writing. A Pathways-RRT seminar offers opportunities for work-in-progress sessions and presentation of research results (poster, oral) with faculty and peer feedback. PGY3/CRY1 Pathways-RRT residents have small grants available to support data collection focusing on answering important questions and to serve as pilot data for future grant applications. The program offers close mentorship and utilizes individualized research plans, collaborative mentoring teams, annual progress reports, surveys of residents and program faculty to evaluate program strengths and weaknesses to foster continuous program improvement. Participants benefit from complementary institutional commitment including the Psychiatry Research Innovations services (a $360,000 per year commitment to provide participants research support including grants administration, research operations, educational workshops, pilot grant opportunities, clinical research support, and biostatistics). The program seeks to prepare residents to compete for post-doctoral research fellowships and grant funding (e.g., NRSA, R21, K) and to prepare them to assume faculty positions at strong academic institutions pursuing research-oriented careers. Thus, the program seeks to address the dearth of physician-scientists in Psychiatry and to produce actionable resident-derived research products that enhance knowledge regarding the causes of mental illness and improve clinical outcomes for patients.