University Of Colorado Denver

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT: Substance use disorders are prevalent, are associated with serious morbidity and are common causes of preventable death. Although providers have at their disposal a number of evidence-based treatments, such treatments tend to be of near-moderate effect size, leaving some patients as non-responders. Work is needed to enhance existing or develop new treatments with higher effects sizes. This mid-career investigator award in patient-oriented research would enable Joseph Sakai, MD release from clinical and administrative duties to allow continued career development activities, to expand his lab’s work on neuromodulation and addiction and to provide high-quality mentorship to early career investigators. Dr. Sakai is involved in several medication trials for the treatment of alcohol use disorder and is contact PI for a UG3 award to test deep brain stimulation for treatment refractory methamphetamine use disorder utilizing a crossover design. This mid-career award, if funded, would allow Dr. Sakai’s lab to test the safety and feasibility of an accelerated transcranial magnetic stimulation treatment protocol for methamphetamine use disorder. In addition, this trial would allow effect size estimation to power a future trial. Each project allows key opportunities for trainee participation and mentorship as described in the application. Dr. Sakai is director of the CU-Pathways resident research track, which seeks to develop research-oriented physician scientists in the Department of Psychiatry. Because of that program’s successes, CU was awarded an R25 to support that track and match 2 residents per year through the NRMP (Sakai MPI). Dr. Sakai has also been very active with the DPRG T32 post-doctoral research fellowship training program which was renewed in 2022 (Sakai is associate director, and is primary mentor for 2 post-docs in that program). Thus, Dr. Sakai has formalized roles within major departmental mentorship programs with a specific focus on physician scientists; all programs emphasize and value recruitment of individuals from backgrounds under-represented in medicine.

NIH Research Projects · FY 2026 · 2023-06

Virus infections of the central nervous system (CNS) are a significant cause of morbidity and mortality worldwide. Proven treatments are limited to only a few viruses and even when treatments exist (e.g. acyclovir for herpes simplex encephalitis) disability and death remain significant. Our knowledge of viral CNS infections, particularly those involving the spinal cord, is limited and serves as a barrier against the development of novel treatments for these devastating diseases. Enteroviruses are an important cause of virus-induced CNS infections. Poliovirus is the most infamous member of the neurotrophic enteroviruses. However, several non- polio human enteroviruses (NPEVs), including EV-D68, also target the CNS. NPEVs are common, causing an estimated 10–15 million symptomatic annual infections in the US alone. Although most of these infections do not result in CNS disease, these viruses can acquire the ability to be neuro-virulent. Recently, large outbreaks of NPEVs have occurred worldwide that have been associated with neurologic disease and these viruses are designated “re-emerging pathogens”. For example, in 2014, the United States experienced an epidemic of acute flaccid myelitis (AFM) cases in children during a nationwide outbreak of previously rare enterovirus D68 (EV- D68) respiratory disease. Approximately 50% of AFM patients had EV-D68 in respiratory secretions. However, EV-D68 was not detected in the cerebrospinal fluid of any patient, preventing the establishment of a causative link between EV-D68 and AFM. We have recently shown that many (including IL/52 and MO/47), but not all (such as CA4231), clinical isolates of EV-D68 from the 2014 outbreak cause neurologic disease in neonatal mice and propose to use this novel model of virus-induced CNS disease to define patterns and mechanisms of EV- D68-induced paralysis. Using chimeric viruses we have demonstrated that the 5’ untranslated region (UTR) and the viral structural proteins VP3 and VP1 are determinants of paralysis. In the proposed studies we will use similar methods to determine which viral sequences are involved in specific mechanisms of paralysis, including neuronal infectivity and apoptosis. An increased knowledge of pathogenic mechanisms that are involved in EV- D68-induced CNS disease will inform the development of antivirals for EV-D68-induced AFM. In addition, by focusing our efforts on host responses to EV-D68 infections our experiments may identify therapeutic strategies that have broad spectrum applicability to additional EVs. Our studies may also have relevance for other viral and non-viral causes of CNS disease.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Heart failure (HF) due to ischemic heart diseases such as myocardial infarction (MI) remains a global health crisis and new therapies to limit the progression to HF after MI are greatly needed. Patient outcomes after MI largely depend on the magnitude, severity, and duration of tissue remodeling - a complex process that involves acute and chronic changes in the structure, function, and cellular makeup of the heart in response to injury. In particular, proper scar formation is required for adequate healing and maintenance of cardiac function. However, the injured heart is predisposed to chronic inflammation and excess scarring, or fibrosis, which promotes cardiac dysfunction, pathological remodeling, and propensity towards HF. It is well recognized that the inflammatory response during post-MI remodeling is a critical determinant of whether scar formation proceeds in a beneficial way to achieve tissue healing or progresses to chronic pathological fibrosis. Inflammation in the post-MI setting is both beneficial and detrimental. For example, acute MI patients given broad-acting anti-inflammatory agents are predisposed to wall rupture due to a muted fibrotic response, highlighting the critical role of inflammatory cells in mediating acute healing through fibroblast activity. Prior work from the proposal PI as a postdoctoral fellow uncovered an unexpected paradigm where activating a subset of innate immune cells, cardiac tissue- resident macrophages (TRMs) expressing the chemokine receptor CX3CR1 (CX3), improved cardiac wound healing and limited fibrosis after MI in a mouse model. This provided proof-of-concept evidence that certain aspects of the inflammatory response can be selectively enhanced to keep post-MI remodeling in balance and improve outcomes. However, the precise cellular signals that drive this pro-healing phenotype in macrophages remain unclear, preventing the development of therapies that harness these beneficial effects of cardiac TRMs. This proposal seeks to address this by elucidating the cellular and molecular mechanisms whereby CX3+ TRMs resolve chronic inflammation and pathological fibrosis in a mouse MI model. To achieve this, we will employ two distinct genetic mouse models to inhibit CX3+ TRMs, genetic macrophage tracking, a well-defined surgical model of MI, and cutting-edge multi-omics, biophysical, and molecular assays of cardiac fibrosis. In Specific Aim 1, we will test the hypothesis that CX3 is required for cardiac TRMs to promote healing post-MI, through attenuating fibroblast expansion and extracellular matrix remodeling. In Specific Aim 2, we leverage a comprehensive spatial transcriptomics and proteomics approach to test the hypothesis that local cardiac microenvironment cues from other subsets of inflammatory macrophages prevent resolution of fibrosis and tissue healing by CX3+ TRMs. Overall our Proposal will determine how CX3+ TRMs act mechanistically to promote myocardial healing and resolve chronic inflammation and fibrosis. These data will reveal potential therapeutic pathways to enhance infarct repair by promoting CX3+ TRM functions. Our long-term goal is to contribute novel insights to better understand and therapeutically modulate cardiac tissue remodeling, thus limiting the progression of HF.

NIH Research Projects · FY 2024 · 2023-06

1 Polycystic ovary syndrome (PCOS), one of the most common endocrinopathies in women, presents with 2 anovulation and hyperandrogenism in adolescence. In addition to reproductive abnormalities, PCOS is 3 frequently associated with obesity and metabolic complications including diabetes, non-alcoholic fatty liver 4 disease, obstructive sleep apnea, hypertension and hyperlipidemia. Additionally, women with PCOS are more 5 likely to suffer from mental health disorders and significant dermatologic manifestations. PCOS is a chronic 6 heterogeneous familial disorder and thus symptoms, signs and co-morbidities at time of diagnosis vary. Despite 7 the high prevalence and gravity of comorbidities associated with PCOS, widely effective therapeutic options are 8 lacking. Recommended therapies for PCOS treatment in women not seeking pregnancy include estrogen 9 containing contraceptives (EC), lifestyle modification, and metformin. Recently there has been a call for more 10 individualized and personalized therapeutic approaches, however there is little biologic basis to recommend one 11 therapy over another. Further, despite the availability of these treatment options for over 40 years, research on 12 their efficacy in youth with PCOS is limited to small studies. Additionally, due to puberty specific hormone 13 changes, adolescents have lower insulin sensitivity than adults. Indeed, a number of studies have established 14 that youth with diabetes or obesity respond less to metformin than adults. Thus, data from medication trials in 15 adult women cannot be applied in youth with PCOS. 16 No mechanism is currently available in the United States to query the early natural history and response to 17 existing medications in a diverse population of youth with PCOS. Using 18 institutions, e found that reproductive abnormalities are more severe in girls without obesity, and metabolic 19 co-morbidities are more common in girls with obesity. Additionally, we and others have found that PCOS 20 presentation, especially as relates to comorbidities, may reflect underling racial and ethnic differences. The 21 overall goal of the project is to describe the presenting reproductive and metabolic phenotypes, natural history, 22 and response to therapy in a large sample of geographically, ethnically and racially diverse youth with PCOS. We 23 hypothesize that presentations of PCOS in adolescence will vary by family history, obesity, and race and ethnicity 24 status, and that unique factors at diagnosis can be used to identify those that respond to EC, metformin or 25 lifestyle therapies. SA1) Describe the presentation and co-morbidities at the time of diagnosis of PCOS in youth 26 in the United States SA2) Determine the response to common medications (lifestyle, EC, metformin). This 27 cohort represents a very diverse population of youth with PCOS, and the database is the first of its kind for youth 28 with PCOS. This work will establish how early phenotypes can inform more individualized approaches toward 29 initial therapy. These efforts are a critical first step towards developing data-informed personalized treatment 30 plans to improve the long-term health of girls with PCOS. pooled retrospective data from 6 w

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Research on the gut microbiome as a target for disease prevention and therapy is an intriguing arena for public health because microbes and their metabolites are modifiable. Observational studies, murine models and a few trials in humans suggest dietary factors exert many of their health effects in the host through modification of the gut microbiome and its associated metabolome. Moreover, early evidence indicates the microbiome and metabolome modify the effects of diet interventions on health outcomes, suggesting the microbiome and metabolome have a role in precision nutrition. However, rigorously controlled feeding trials in humans are still needed to determine the effects of whole diet inverventions on the microbiome and metabolome, and to test if the microbiome and metabolome modify or mediate the effects of diet on cardiovascular risk factors, including blood pressure. Of particular interest are the effects of dietary patterns and level of sodium (Na+) intake. The primary objective of this study is to examine the effects of the American Heart Association recommended Dietary Approaches to Stop Hypertension (DASH) diet (compared to a typical U.S. diet) and lower dietary Na+ intake vs. higher dietary Na+ intake on the gut microbiome and the untargeted and targeted metabolome— including short chain fatty acids—in a multi-racial cohort of adults with type 2 diabetes enrolled in the DASH4D trial – a recently funded randomized, cross-over, isocaloric controlled feeding trial. A secondary objective is to explore if the gut microbiota and their metabolites modify and/or mediate the effects of diet patterns and Na+ on blood pressure, thusly informing precision nutrition. We will also examine if effects vary by sex. In particular, we propose to perform whole genome shotgun metagenomics, high-throughput metabolomics profiling, and targeted quantification of short chain fatty acid metabolites. We will jointly investigate the microbiome and metabolome measured in stool and blood collected before and after each of the diet periods in the feeding trial. Dr. Mueller (PI) will carry out this research with an outstanding group of interdisciplinary co-investigators in the collaborative and eminent environments of the Johns Hopkins Welch Center for Prevention, Epidemiology and Clinical Research and the Bloomberg School of Public Health. Dr. Mueller’s co-investigators have complementary expertise in feeding trials (Appel), -omic statistics (Zhao), bioinformatics (Debelius, Bittinger), metabolomics (Rebholz), and short chain fatty acids (Pluznick). With the support of Dr. Mueller’s research team, he is well positioned to complete the proposed activities. The findings have great potential to: (a) identify objective measures of adherence to the DASH diet and lower dietary Na+ intake; (b) reveal novel mechanisms underlying the BP effects of dietary patterns and Na+ intake; (c) offer new disease prevention strategies and therapeutic possibilities and; (d) inform use of the microbiome-metabolome nexus for precision nutrition.

NIH Research Projects · FY 2025 · 2023-06

Summary Individuals trained in both data science and genomics are essential to fully realizing the plethora of data at the intersection of genomics and biomedicine. Additionally, diversity in the genomics workforce is necessary to support creativity, the study of conditions in underrepresented groups, and ensure that research performed is generalizable and beneficial to all people. Here, we propose the Pathways in Genomic Data Science (PATH-GDS) research experience program for MS Statistics and MS Applied Mathematics students within the Department of Mathematical and Statistical Sciences at the University of Colorado Denver (CU Denver). CU Denver is uniquely positioned with two campuses: the Downtown Campus with undergraduate programs and close proximity to diverse recruiting institutions, and the Anschutz Medical Campus with world renowned biomedical and human genomics research. In PATH-GDS we will: (1) recruit scholars from diverse groups underrepresented in biomedical and genomics research; (2) train quantitative MS students from diverse backgrounds in genomics research and career skills; and (3) create, support, and retain a diverse data science genomics community. In doing so, we will build and support a diverse data science genomics community within Colorado, the Mountain West, and beyond.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY The diversity of the United States population is not reflected in its biomedical sciences workforce, with the greatest disparities at the highest levels of degree attainment and in careers that require advanced degrees. Institutions of higher education have both the capacity and the imperative to address the structural barriers that create inequitable outcomes in degree attainment for underrepresented populations, both at the undergraduate and graduate levels of education. The University of Colorado Denver (CU Denver) is unique and uniquely positioned to meet this goal. CU Denver (comprised of the downtown Denver campus and the Anschutz Medical Campus) is a “Doctoral University: Higher Research Funding” with a funding portfolio of $790 million and a combined enrollment of 19,395 students. It is the only public urban research university in the State of Colorado and the most diverse institution in the CU system: on the Denver campus, at least 58% of incoming first-year students identify as students of color and 43% as underrepresented minority students. CU Denver's commitment to equity, lifelong learning, innovation, research excellence, and community care underlie its record of investment in undergraduate research and improving student success outcomes, including the timely graduation of undergraduate students and their transition into doctoral programs in biomedical sciences. The purpose of the CU Denver Maximizing Access to Research Careers program proposed here (MARC at CU Denver) is to continue this record of excellence and further contribute to the development of a diverse and inclusive national biomedical research workforce by providing underrepresented, honors-eligible undergraduate science majors with the courses, structured training activities, mentoring, and authentic research experiences necessary to transition successfully into research-focused biomedical doctoral programs. With prior funding and institutional support, CU Denver has spent nearly a decade developing, implementing, evaluating, and refining a successful training model that builds trainees' science knowledge and scientific thinking skills, research experience, communication and networking skills, sense of belonging and wellness skills, and career development skills. With continued institutional support and a record of success with a similar program, a new MARC at CU Denver program will support 30 trainees over five years such that 90% or more will complete their baccalaureate degrees in a biomedical science related field at CU Denver (80% or more in two years after joining the program) and 60% or more will matriculate into doctoral programs in biomedical science within three years of graduating, with an 80% doctoral program completion rate. The success of MARC at CU Denver is bolstered through institutional support for a base-building “pre-MARC” program for first and second-year students, scholar wellness and resiliency training and support, and continued external program evaluation. The scope of MARC at CU Denver is broadened through direct institutional support for additional senior CU Denver MARC Affiliate Scholars.

NIH Research Projects · FY 2024 · 2023-06

PROJECT SUMMARY Diastolic dysfunction (DD), characterized by impaired left ventricular compliance and relaxation, is associated with increased risk of developing heart failure with preserved ejection fraction (HFpEF), a devastating syndrome with poor prognosis for which there currently exist limited therapeutic interventions. Dynamic acetylation of histones represents a critical component of chromatin-dependent signal transduction involved in the activation of cardiac fibroblasts (CFs) and increased extracellular matrix deposition, leading to progressive DD and development of HFpEF. These processes are largely regulated by histone deacetylases (HDACs), a family of epigenetic regulatory enzymes whose pharmacological inhibition is cardioprotective in the setting of DD; however, little is known regarding the HDAC isoform specificity and molecular mechanisms mediating this protection. This Pathway to Independence award will leverage innovative small molecule inhibitors, genetics- based strategies for cell type-specific gene ablation, and the integration of multifaceted state-of-the-art epigenomic and bioinformatics techniques to examine the cell type- and isoform-specificity of HDAC inhibition (Aims 1 and 2), and therapeutic potential of inhibition of a novel glycan binding protein (Aim 3), in myofibroblast activation, cardiac fibrosis and DD. In Aims 1 and 2, the applicant will train with co-mentors and advisors in the K99 phase in a single-cell, genome-wide next generation sequencing technology that characterizes chromatin architecture, a flow cytometry-based technique for characterizing inflammatory cells, an integrated approach to transcriptomics and proteomics analyses in primary human CFs, and a genetics-based approach for cell type- specific gene ablation, all with the overall goal of defining the cellular specificity and molecular mechanisms mediating the cardioprotective properties of HDAC inhibition. In the R00 phase described in Aim 3, the applicant will utilize the skills acquired in the K99 phase to investigate the role and therapeutic potential of inhibiting the glycan-binding protein Galectin-1, recently discovered to be significantly altered in the CF population of mice with DD and subjected to HDAC inhibition, in myofibroblast activation, cardiac remodeling, and the progression to HFpEF. The applicant possesses extensive prior knowledge in epigenetics, CF biology, and the pathophysiology of DD and fibrotic remodeling. Furthermore, the mentorship team consists of internationally recognized leaders in epigenetic regulation of cardiovascular disease, clinical HFpEF, murine models of HF, and emerging bioinformatics technologies. The environment at the University of Colorado Anschutz Medical Campus is exemplary for collaborative and innovative research, with an excellent infrastructure including a human heart biorepository and outstanding core facilities. In summary, the exceptional mentoring team and institutional environment will provide a solid foundation for the applicant’s development into an independent investigator. Moreover, this innovative approach offers the exciting potential to contribute to the development of desperately needed novel therapeutic strategies for the treatment of heart failure.

NIH Research Projects · FY 2024 · 2023-06

Project Summary The proposed studies will examine mechanisms by which advanced age increases intestinal permeability and neuroinflammation after burn injury using a clinically relevant mouse model. Regardless of age, most burn patients do not die from primary injuries, but rather from complications, such as sepsis. Further, aged burn patients often experience greater neurological impairments, which may stem from heightened neuroinflammation. Clinical and experimental evidence reveal that healthy aged subjects are in an elevated basal inflammatory state, referred to as “inflammaging,” which can contribute to deficits in tissue injury and repair. We and others believe that inflammaging is caused by translocation of bacterial products from the intestinal lumen and that exposure to these products triggers the production of pro-inflammatory cytokines and chemokines, including tumor necrosis factor alpha (TNF), interleukin (IL)-1β, IL-6, and C-C Motif Chemokine Ligand 2 (CCL2). Novel preliminary data in our clinically-relevant murine model of scald burn injury confirm that aged mice who sustain a burn injury have heightened circulating levels of danger-associated molecular patterns (DAMPs), and a greater breach in intestinal epithelial barrier integrity that coincides with an increase in markers of neuroinflammation. Both neuroinflammation and burn injury in the aged population have been correlated with breaches in the blood brain barrier, delirium, and other signs of cognitive decline. From these observations, we hypothesize that post-burn gut leakiness seen in aged mice is driven by excessive IEC death, ISC dysfunction, and reduced IEC proliferation. Additionally, these changes in the gut lead to leakiness of the blood brain barrier and neuroinflammation. To test this hypothesis, in Aim 1, we will investigate the mechanisms behind gut leakiness in young and aged sham and burn-injured mice by identifying intestinal epithelial cell apoptosis/necroptosis, epithelial cell proliferation, and intestinal stem cell markers in vivo and in vitro utilizing whole tissue, isolated epithelium, and intestinal organoids, along with measuring blood-borne gut-derived bacteria and bacterial cell wall components. In Aim 2, we will examine blood brain barrier integrity in young and aged sham and burn-injured mice using multiple measures of barrier permeability. Further, we will examine the levels of pro-inflammatory cytokines and chemokines, and microglial and astrocyte activation within brain regions. Finally, we will determine if limiting intestinal barrier damage after burn injury reduces neuroinflammatory markers in the brain. These studies will expand our understanding of how advanced age alters the gut in the context of burn injury and the impact of intestinal permeability on neuroinflammation. It is our hope that our work will lead to the development of novel therapies to treat the excessive inflammatory response and consequences of that inflammation in burn patients of all ages.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY The human skin is the largest organ of the body. This physical barrier and the skin's innate immunity are an essential first defense against pathogens. Staphylococcus aureus is an opportunistic pathogen and the dominant microorganism of soft skin and tissue infections. These infections can quickly develop into systemic infections and worsen health outcomes. Increasingly, antibiotic-resistant epidemic S. aureus strains (e.g. methicillin-resistant S. aureus, MRSA) complicate treatment of these infections in the clinical setting. Although skin colonization increases risk of a MRSA infection, studying this aspect of host-pathogen interactions has been challenging. Current animal models lack the acidity, skin structure, and eccrine gland distribution of human skin and are not accessible to many research groups. Additionally, in vitro testing with media mimicking the human skin surface have not been published. To address these gaps, I developed an in vitro media that incorporates the metabolites, ions, and pH that MRSA would encounter on the human skin. I performed RNA-seq of MRSA grown in these conditions and compared these results to a previous RNA-seq experiment of MRSA inoculated on mouse skin. In both datasets, genes for metabolizing urea, derived from sweat glands; and urocanic acid, derived from the natural moisturizing factor, were significantly upregulated. I hypothesize that the metabolism of urea and urocanic acid contributes to S. aureus pH homeostasis and growth, respectively, on the skin. This hypothesis will be tested in the following aims: Aim 1: Ascertain the role of urease and related functions in MRSA growth and pH homeostasis in human skin-like in vitro models. Genetic and biochemical approaches will be used in an in vitro media and a differentiated keratinocyte model to investigate the role of urease, contribution of urea and nickel transport to urease function, and therapeutic approaches to inhibit urease activity Aim 2: Investigate the role of urocanic acid metabolism in MRSA skin colonization. Genetic and biochemical approaches will be used in the previously mentioned models to validate the predicted encoded functions of this pathway, investigate substrate specificity of the pathway, and better understand regulatory control. Epidemiology of pathway expression will be assessed by Western Blot analysis of clinical S. aureus skin isolates from atopic dermatitis patients and healthy controls. These studies will provide a foundation for future research of the skin microbiota, expand our understanding of MRSA physiology on the skin, and identify potential targets for future therapeutic development.

NIH Research Projects · FY 2026 · 2023-05

Summary Rhabdomyosarcoma (RMS) accounts for 3-4% of all pediatric cancers, with less than a 30% overall 5-year survival rate for children diagnosed with metastatic RMS. Sarcoma patients also experience higher rates of morbidity and mortality than other cancer patients, and this is particularly evident in children. As a result of their therapies, 42% of childhood cancer survivors experience severe, disabling, or life threatening conditions, including secondary tumors. Thus, there is clearly a need to develop new, more targeted treatment strategies for pediatric tumors such as RMS; treatments that inhibit tumor progression yet confer limited side effects. In many cancers, embryonic programs, including the acquisition of stem/progenitor states, are instituted to contribute to tumor progression. Such reinstatement or retention of developmental programs may be a key driving factor in Embryonal Rhabdomyosarcoma (ERMS, which are generally fusion negative and also referred to as FN-RMS). In RMS, high expression of myogenic-lineage transcription factors (TF), MYOD1 and MYOG, is observed. However, despite high expression of these TFs, RMS cells fail to differentiate. Instead, these TFs drive the RMS malignant phenotype, since knockout of these MRFs results in lethality to the RMS cell. Thus, a key unanswered question in the field is why and how the myogenic regulatory factors (MRFs) depart from their canonical roles as drivers of muscle differentiation to instead maintain RMS cells in an undifferentiated state. In this proposal, we are examining whether SIX1, a critical TF that regulates muscle development, is responsible for globally altering the function of MRFs. Our data show that elevated SIX1 expression promotes FN-RMS progression and growth by promoting an RMS progenitor-like state. Intriguingly, SIX1 knockdown induces a muscle differentiation signature, concomitant with re-localization of key MRFs genome-wide. These data suggest that in FN-RMS, SIX1 overexpression alters MRF function, promoting an RMS progenitor-like phenotype and enhancing tumor growth and progression. Thus, in this proposal we will test the following hypothesis: SIX1 and its obligate cofactors EYA2/3 cooperatively drive the progression of FN-RMS by altering the genomic landscape, causing MRFs to favor growth over differentiation. Since SIX1 expression in differentiated tissues is low, targeting it may therefore be a means to inhibit FN-RMS with limited side effects to untransformed cells where SIX1 is dispensable. Specific aims are as follows: 1) To determine the molecular mechanism by which SIX1 serves as a master regulator of the muscle progenitor vs differentiated state in development and in RMS. 2) To identify the critical SIX1 cofactors (with a focus on EYA proteins) that, when targeted, can induce differentiation and inhibit RMS growth. This work will take advantage of normal developmental regulatory mechanisms to inhibit the tumor, and thus may have limited toxicity due to the paucity of SIX/EYA expression in adult tissue. Our ability to combine zebrafish and human models will maximize the benefits of each model system to rapidly and inexpensively identify means to inhibit RMS growth and progression. Understanding the mechanisms by which RMS cells are trapped in an early developmental state may therefore lead to novel means to target the disease.

NIH Research Projects · FY 2026 · 2023-05

The rapidly changing landscape of cannabis policy in the United States is well known, but often overlooked is that legalization has made high-potency products more available. State-regulated cannabis markets offer access to cannabis products, such as concentrates with THC potencies (70-90%) that far exceed cannabis that has been sold on the black market over the last 20 years (4-12% THC potency). State reporting suggests that concentrates are commonly used and quickly rising in popularity, with increases in 2014-2019 sales (409%) far outpacing that of flower (80% increase) and edibles (234% increase). Recent data indicate that concentrate users exhibit higher plasma THC levels than flower users and a large body of research has demonstrated dose-response relationships between increasing cannabis consumption and poor mental health outcomes. Given the increasing prevalence of concentrate use, it is imperative to clearly understand how these increasingly popular, high-potency cannabis products affect mental health and psychosocial functioning. This project leverages an ongoing, longitudinal twin study, with participants assessed approximately every 5 years since adolescence. Thus, this project is a unique opportunity to rigorously test critical hypotheses about how high-potency concentrates affect psychopathology and psychosocial functioning. The Aims of this research are: Aim 1: Examine how the use of different cannabis products relates to cannabinoid exposure (e.g., THC). We will test whether the use of high-potency cannabis concentrates, compared to flower products, is associated with elevated cannabinoid exposure. We hypothesize that associations will not be explained by familial confounds (via co-twin comparisons) and individual differences (via repeated assessments). Alternatively, it is possible that concentrate users titrate to reach the same effect as flower users. Aim 2: Examine how the use of different cannabis products relates to mental health and psychosocial functioning. It is plausible that individuals vary in their propensity to use high-potency concentrates. Thus, we will examine how use of high-potency concentrates relate to mental health while accounting for familial and individual confounds, like genetics and prior substance use. We hypothesize that twins who uses more high- potency concentrates will tend to have greater levels of depression, anxiety, and impulsivity and psychosocial dysfunction compared to their co-twins who use flower or who use high-potency concentrates less frequently. Aim 3: Test whether different levels of cannabinoid exposure are associated with mental health and psychosocial outcomes. We will test how different levels of cannabinoid exposure (i.e., THC-COOH concentrations in whole blood) associate with depression, anxiety, and impulsivity as well as psychosocial functioning. That is, we will directly test the hypothesis that cannabinoid exposure is associated with mental health and psychosocial functioning. We hypothesize that higher levels of cannabinoid exposure will be associated with worse symptoms of mental health and psychosocial functioning.

NIH Research Projects · FY 2025 · 2023-05

Abstract There is an urgent need to understand the factors determining female reproductive longevity. Female reproductive aging is characterized by a significant decline in reproductive function due to the sequential decrease in the number and quality of ovarian follicles that constitute the finite ovarian reserve. The rete ovarii (RO) is a conserved epithelial structure divided into 3 regions, the intraovarian rete (IOR), located within the ovary, the extraovarian rete (EOR), located in the periovarian tissue, and the connecting rete (CR), which links the EOR and IOR. The RO has been depicted for decades in anatomical context, and is a possible contributor to gynecological disease, yet its function has never been thoroughly investigated. The reproductive longevity of females is determined by three main factors: 1) the size of the initial primordial follicle pool, 2) the rate of ovulation and atresia during the fertile window, and 3) perturbations, such as changes in metabolic status or hormone levels, which affect ovarian function and follicle growth. The objective of this application is to elucidate the contribution of the RO to each of these determining factors. To allow visualization and functional investigation of the ovary in situ, I developed tissue clearing and 3D imaging methods that suggest a role for the rete in the assembly of ovarian cells into ovigorous cords and subsequent specification of medullary and cortical domains. I found that the RO remains closely associated with the ovary throughout adulthood, and preliminary ovary transplantation experiments suggest that the host EOR/CR reestablishes a connection to the IOR of the grafted ovary. Our transcriptomic data of the RO shows enrichment of secretory and metabolic pathways consistent with the finding that Dextran injected into the EOR is transported to the ovary. These data have led to the central hypothesis that the RO plays critical roles in the establishment of the ovarian reserve and in the regulation of ovarian follicle growth in response to physiological cues. This hypothesis will be tested by pursuing three specific aims: (1) (K99) Characterize the contribution of the RO to the development of the ovary and the establishment of the ovarian reserve; (2) (K99/R00) Define the functional differences between EOR and IOR in adult homeostasis and (3) (R00) Investigate the changes of the RO in response to physiological cues. Discovering a role for the rete ovarii in the establishment of the ovarian reserve and in the regulation of adult follicle growth under homeostatic or perturbed conditions will situate the RO as a new candidate in the search for determinants of female fertility and reproductive longevity. In addition, the approaches I develop will be useful in future investigations of interactions between the ovary and other extrinsic tissues. Pursuit of these aims requires training in single-cell RNA sequencing, mouse surgery and virally-mediated approaches for manipulating gene expression. I have assembled an advisory committee chaired by Dr. Capel, and we designed a training plan that will provide me with the skills required to run a productive, independent developmental and reproductive biology laboratory.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY / ABSTRACT Advances in sequencing technologies provide new opportunities to interrogate biological systems from multiple perspectives. However, the introduction of new technologies highlights a problem many researchers face: missing data. Missing observations across technologies and biological states is a frequently observed problem in the field of computational biology. This missingness can be a result of limitations in the technology, the rarity of a biological state, or because the technology has not been widely adopted. While one technology may have high sparsity in biological observations, there is an opportunity to leverage existing, complementary data from an established technology to impute the missing biological observations. We address these issues by utilizing new methodological advances in machine learning, primarily focusing on domain adaptation techniques. These techniques learn patterns in one dataset that can be adapted to another dataset, enabling cross-technology information sharing. Our proposal introduces a general framework in which domain adaptation techniques can be used to unite an emerging technology with a different, but technology. To highlight the broad utility of this approach, we apply this model to three biomedical applications: 1) Predict cell-type-specific perturbation response in rheumatoid arthritis; 2) Predict tissue-of-origin from cell-free DNA (cfDNA); 3) Predict progenitor-specific gene signatures from cell-free DNA in acute myeloid leukemia (AML). The proposed aims not only unite existing and emerging sequencing technologies, but enable the discovery of new biology that is difficult or infeasible to directly observe. The research proposed builds on my experience in using statistical approaches for transcriptomic data. During the K99 phase I will require further training from my mentoring team in deep generative modeling (Dr. Casey Greene), modeling of single-cell data (Dr. Fan Zhang), and modeling of cfDNA and chromatin accessibility (Dr. Srinivas Ramachandran). The research will be conducted at the University of Colorado, Anschutz Medical Campus, in the Center for Health AI. In this institution, I will have access to the Colorado Clinical and Translational Sciences Institute and the RNA Bioscience Initiative, which provide resources for building an interdisciplinary and translational research program. With this training and available institutional resources, I will have a solid foundation on which to build an independent research program focused on domain adaptation applications for high-throughput sequencing technologies.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT Recent technological breakthroughs have enabled the generation of clinical, environmental, and multi-omics data at an unprecedented scale, providing a complete profile of the patient for individualized disease diagnosis, prog- nosis, and treatment. However, the precision medicine approach is yet to realize its potential in most multi-factorial diseases, for which their highly polygenic nature, as well as phenotypic and genetic heterogeneity, complicate the identification of disease-associated cell type-specific transcriptional mechanisms. A better characterization of this heterogeneity and an interpretable prediction of individuals at high risk of disease are crucial steps to deliver the promises of precision medicine. In this context, polygenic risk scores (PRS) are likely to play a crucial role in precision medicine for disease-risk prediction. However, it has been argued that PRS might accentuate dispari- ties among non-European ancestries and have low stability at individual-level predictions, probably due to greater underlying complexity in disease etiology that is not captured in a single score. Current efforts to mitigate health disparities involve recruiting individuals from different population ancestries. However, if the underlying biological complexity of disease etiology remains unaccounted, risk stratification methods will continue to be limited. The goal of this project is to develop machine learning methods to advance key computational aspects of precision medicine. In the first aim, an unsupervised method will be applied across large amounts of genetic studies to detect gene sets associated with multiple human traits, which will also identify environmental risk factors. In the second aim, new computational approaches will be developed to learn gene co-expression patterns optimized for a better understanding of transcriptional mechanisms linked to complex traits and their therapeutical modalities. This will detect gene modules (i.e., genes with similar expression profiles across the same cell types) with complex gene relationships, and the approach will be validated by predicting known FDA-approved drug-disease links. Finally, the outcomes of these aims will inform a gene module-based polygenic risk score for accurate and robust disease-risk stratification that will be portable across different population ancestries. Although the methods will be initially applied to asthma, they are clearly extendable to other common diseases as well. For the K99 phase of this project, the mentorship team's expertise covers all key areas of precision medicine, including computational genetics, systems biology, environmental exposure studies, pharmacology, and trans- lational medicine. Mentors and advisors are directly involved in precision medicine initiatives to enhance both scientific discovery and its implementation in clinical care. For the R00 phase and beyond, all the conceptual and methodological expertise previously learned will prepare the applicant for an independent research career in computational methods development applied to precision medicine. The Perelman School of Medicine at the University of Pennsylvania, consistently ranked among the top research medical schools, represents the ideal environment for this highly collaborative project.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Synaptic plasticity is well accepted as the basis of behavioral adjustability in the face of a constantly changing environment. Our lived experience is transmitted to our brain as electrical impulses along axons. Oligodendrocytes (OLs) increase the rate at which these electrical impulses are transmitted by insulating axons with myelin sheaths. Surprisingly, motor learning, sensory stimulation, and social enrichment induce the differentiation of precursor cells into myelinating OLs resulting in quantifiable structural changes in white matter. These findings point to myelin plasticity as a concurrent, and equally important contributor to the adaptability of neural circuits. However, the molecular and cellular mechanisms underlying myelin plasticity are not well understood. Single OLs can give rise to sheaths of different lengths and thicknesses to accommodate the needs of diverse axons. These observations suggest a local and independent regulation of myelination at the level of individual sheaths. How do sheaths assess the needs of specific axons? Action potentials cause axons to, not only release vesicles at their terminal ends, but also along their shafts. Our lab and others have shown that axons signal to myelin sheaths via these alternative release sites and that myelin sheaths express the canonical post-synaptic factors required to interpret these signals. These data suggest that the use of a shared transmission machinery enables synaptic and myelin plasticities to occur in parallel as a response to the same stimulus. While some components of axo-myelin communication have been elucidated, the intracellular mechanisms bridging signal receipt to myelin production remain unknown. In dendrites, the localization of mRNA transcripts and ribosomes to individual spines support their rapid, tailored adaptive responses. Similarly, diverse groups of mRNAs, along with ribosomes localize to myelin sheaths raising the possibility that local RNA translation underlies the ability of individual OL sheaths to fine-tune their responses to signals from various axons. Due to the dynamic nature of RNA translation, it would be best understood if studied in vivo. However, limitations in technological approaches stood in the way for decades. Using diverse transgene expression systems, protein photoconversion technology, and my expertise with 2-photon laser severing, I will determine if local translation of myelin-resident transcripts occurs in zebrafish. Additionally, I will investigate whether the myelin localization of an enriched group of transcripts we identified contributes to myelin plasticity. To accomplish this, I will create a loss-of-function mutation of Khdrbs1, an RNA binding protein predicted to bind to members of this enriched group. Finally, I will test if manipulating neuronal activity alters the translation of targeted myelin resident mRNAs. This work will add to our understanding of how axo-myelin exchanges impact the efficiency of neuronal circuits by providing new insights into the kinetics of local translation in vivo.

NIH Research Projects · FY 2026 · 2023-05

The goals of this study are to understand how inhibiting DNA-PK activity 1) induces and increases tumor antigen/neoantigen expression in weakly immunogenic tumors and 2) contributes to maintaining dendritic cells and myeloid cells in a T cell-activating state. These goals are motivated by exciting clinical results which demonstrate that T cell-based immunotherapies can mediate tumor regression but also because durable responses to current therapies are observed in only a subset of patients and against a limited number of cancers. Favorable responses to immunotherapies correlate with levels of neoantigens and changes in the tumor-reactive TCR repertory. Cancers can evade T cell detection by downregulating major histocompatibility complex (MHC I) and antigen expression. We screened ~2,500 compounds for the ability to selectively regulate the expression of various tumor-associated antigens (TAAs) and increase MHC I expression. Among the most effective drugs were DNA-PK inhibitors. Our preliminary studies indicate that DNA-PK inhibition increases and diversifies the expression of various TAAs and neoantigens in melanoma at the transcriptional level leading to increased protein expression. Further, reduced DNA-PK levels or DNA-PK mutations in patient samples are associated with increased tumor infiltrating lymphocytes (TIL) and tumor mutation burden. The overarching hypothesis is that DNA-PK plays a novel role as a transcriptional regulator that modifies the expression of melanoma tumor antigens, including neoantigens, and thereby increases the diversity, frequency, and activity of tumor-reactive T cells. We further postulate that inhibiting DNA-PK activity in DCs and myeloid cells reduces their propensity to enter a T cell-suppressive state. These hypotheses will be addressed through the following aims. In Aim 1, we will determine the molecular mechanisms by which DNA- PKcs regulates tumor antigen expression by determining the role of kinase activity, identifying the DNA-PK substrate(s) that contribute to its repressive function, and to define the roles that the individual DNA-PK subunits play in transcriptional regulation. Aim 2 will ascertain the in vivo impact that DNA-PK inhibition has on increasing and diversifying the tumor-reactive T cell repertoire. We seek to demonstrate that DNA-PK inhibition treatment increases the number and activity of neoantigen-reactive T cells. Aim 3 will determine the impact that inhibiting DNA-PK has on inducing and sustaining stimulatory signals on tumor derived dendritic and myeloid cells. We will determine how DNA-PK inhibition prevents DCs and myeloid cells from a entering a T cell-suppressive state. The proposed studies are significant as they will offer molecular and cellular insights as to how DNA-PK activity contributes to tumor immunogenicity and exploit these insights to develop more reliable biomarkers and effective therapies against weakly immunogenic tumors.

NIH Research Projects · FY 2025 · 2023-05

Abstract The overall goals of this K24 renewal application are to continue to provide Maryam Asgari MD, MPH with protected time to serve as a mentor to junior clinical investigators, and to support new scientific aims that will build upon Dr. Asgari's established work on patient-oriented research in skin diseases. Dr. Asgari is a nationally recognized dermatoepidemiologist and Mohs surgeon and the Director of the Patient-Oriented Research in the Epidemiology of Skin Diseases (PORES) unit at Harvard Medical School. She is the principal investigator of several large patient-oriented research studies, including two NIH-funded R01s examining genetic and environmental factors impacting the development of skin cancer. The parent K24 award has allowed Dr. Asgari the protected time to successfully mentor over 30 trainees ranging from medical students to junior faculty. This K24 renewal award would allow her to 1) continue mentoring junior investigators, particularly NIH K23 awardees, 2) pursue new research in cutaneous carcinogenesis with a renewed focus on epigenomic changes in carcinogenesis and the role of dietary supplements in keratinocyte carcinoma risk, and 3) establish a new research direction focused on shared decision making in skin cancer care. The research plan (Aim 1) builds on an ongoing NIAMS- and NCI-funded portfolio of projects led by Dr. Asgari. The proposed new aims will take advantage of existing data from these R01 awards and allow mentees the ability to use existing datasets to examine novel topics in keratinocyte carcinogenesis. This renewal will be an important catalyst for Dr. Asgari’s professional growth by enabling her to learn and conduct innovative patient-facing research involving shared decision making in keratinocyte carcinoma management. The mentoring plan (Aim 2) in this continuation period will allow for the implementation of a structured mentoring program tailored at the individual level to engage mentees using a formal individual development plan. Mentoring outcomes will be assessed using a validated tool for anonymous feedback from mentees, and mentoring skills will be refined by utilizing a mentoring coach. Dr. Asgari will continue her successful trajectory of providing direct mentorship to dermatology residents, research fellows, and junior faculty at her institution. Her plan for the next 5 years is to leverage her efforts for career development of patient-oriented researchers at the national level. This K24 renewal will be an important catalyst for Dr. Asgari’s professional growth by enabling her to conduct innovative research and mentor new investigators on performing patient-oriented dermatologic research. The mentoring and research activities in this plan will open new opportunities for a long-term professional trajectory that would not be attainable without a renewal period. By supporting Dr. Asgari’s efforts in mentoring, the award is an excellent investment in the junior investigators at her institution.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY. Down syndrome (DS), caused by Trisomy 21 (T21), occurs in ~1 in 700 live births, making the most commonly occurring chromosomal abnormality. Individuals with DS experience a unique disease spectrum, whereby they are protected from some conditions, including solid tumors, and predisposed to others, such as Alzheimer’s disease and autoimmunity. Among the conditions more common in people with DS are gastrointestinal (GI) abnormalities, including esophageal motility disorders, gastro-esophageal reflux, irritable bowel syndrome, small bowel motility disorders, colonic dysmotility, slow transit constipation, and others. However, the molecular mechanisms underlying these conditions remain unclear, creating challenges for their clinical management. Recent work has established that many of these conditions can be caused by damage to the enteric nervous system (ENS). Furthermore, a mouse model of DS was recently shown to have fewer ENS neurons than its wild- type counterparts. Here, we propose that progressive injury to the enteric nervous system (ENS) drives GI motor and sensory abnormalities in DS. The transformative hypothesis of this proposal is that progressive injury to the ENS drives colonic secreto-motor and permeability (SMP) abnormalities in DS leading clinically to chronic constipation. This proposal could illuminate novel aspects of the pathophysiology of GI diseases, which affect more than 50% of individuals with DS. To address these key research gaps and define the mechanisms underlying ENS dysfunction in DS-associated GI disease, we propose a two-part approach: deep-phenotyping in a cohort study of individuals with DS and cause-effect animal research using mouse models of DS. Our Specific Aims are: Specific Aim 1: To expand our ongoing pan-omics cohort study to define associations between markers of inflammation, metabolic dysregulation, and altered GI microbiota, with chronic constipation. Specific Aim 2: To determine the effects of experimental colonic inflammation and microbiome manipulation in a murine model of DS to define the contributions of alterations in inflammation, metabolism, and the microbiome to colonic function. Together, these efforts will not only define the role of ENS dysfunction in key biological and clinical aspects of DS, but also provide the rationale and data to justify the development ENS-based therapies to serve this population by decreasing neuro-intestinal disease.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY: Identification and treatment of risk factors that accelerate Alzheimer's disease (AD) are essential to slowing disease progression. Alphaherpesviruses (herpes simplex virus type 1 [HSV-1], varicella zoster virus [VZV]) are potential accelerators of AD because they increase dementia risk and produce similar pathologies, including amyloid, neuroinflammation, neurodegeneration, and cognitive impairment1-3. In parallel literature, early AD is characterized by smell loss4,5, amyloid deposition in olfactory epithelium (OE)6,7, and olfactory sensory neuron (OSN) dysfunction (reviewed in8). Because sniff-induced gamma () oscillations generated in olfactory bulb (OB) are directionally coupled to the hippocampus9-12, smell loss would result in decreased hippocampal  oscillations that have been postulated to lead to neurodegeneration and cognitive decline.14-16 Because alphaherpesviruses infect and reactivate in the nasal cavity, alphaherpesvirus disruption of olfactory pathways may accelerate AD. Our preliminary data show: (1) compared to controls, OB and olfactory tract (OT) from familial AD (FAD) subjects have upregulation of viral and inflammatory transcriptional pathways , confirmed at the protein level; (2) VZV immediate early protein 62 was detected in serum of 2 of 3 AD subjects and in 0/4 controls; (3) HSV-1- and VZV-infected human OE cultures (OECs) contain amyloid and increased OSN differentiation; and, (4) intranasal HSV-1-infected 5xFAD mice have increased OE amyloid and decreased olfaction compared to uninfected or pre-inoculation controls, respectively. Taken together, we hypothesize that alphaherpesvirus infection of the OE contributes to pathological processes within the olfactory system and hippocampus, thereby accelerating disease. To test this hypothesis, we will: (Aim 1) identify gene hippocampus, (Aim 2) determine whether infection of human OE with VZV and HSV-1 ex vivo elicits amyloid production and loss of odorant responsiveness, recapitulating smell loss in AD; and (Aim 3) test whether HSV- 1 worsens olfactory dysfunction in 5xFAD mice, accelerating the AD phenotype; specifically, we will test whether HSV-1-induced pathology and functional changes are diminished by optogenetic stimulation of mitral/tufted cells in the  frequency range and if entrainment of hippocampal  oscillations can prevent cellular and behavioral deficits elicited by HSV- 1. Understanding how viruses interact with the aging olfactory system, as well as with individuals who overexpress amyloidogenic peptides (FAD), to accelerate AD will identify potential biomarkers and therapies (e.g. vaccines or antiviral agents) that may slow or halt progression to clinical dementia, disability, and death. expression/pathways supportive of virus nfection in OE, OB, OT, entorhinal cortex, and biological fluids of FAD and sporadic AD (SAD) subjects; i

NIH Research Projects · FY 2024 · 2023-05

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) results from autoimmune destruction of pancreatic β-cells. Potential therapeutic approaches to curtail or reverse the loss of β-cells include immune modulation, β-cell regeneration, or combinations thereof. There has been substantial preclinical development of therapies that stimulate human β- cell replication. However, absolute proliferation rates induced by such drugs remain relatively low, and it is unclear whether current approaches will sufficiently expand β-cell numbers to control blood glucose without exogenous insulin. Here, we build upon emerging evidence that cell metabolism supports proliferation to identify and exploit metabolic bottlenecks that limit therapeutic β-cell expansion. Proliferating cells are known to activate metabolic programs that produce cellular building blocks and generate signals that support cell division. In preliminary data, we found that the metabolic enzyme ATP-citrate lyase (Acly) is required for expansion of transformed β-cells. Other groups have shown that pancreatic deletion of Acly results in smaller islets, consistent with a role for this enzyme in β-cell proliferation. Acly acts upon citrate to produce acetyl-CoA, which is a substrate for lipid synthesis and histone acetylation. In this way, Acly couples mitochondrial metabolism to production of cellular building blocks and signaling via the epigenome. In β-cells, Acly deletion results in downregulation of lipid synthesis genes concomitant with reduced histone acetylation of their associated regulatory elements, suggesting Acly regulates lipid synthesis through dual metabolic and epigenetic mechanisms. Together, these observations build the model that Acly produces both anabolic and signaling metabolites that support β-cell proliferation. We found that Acly is phosphorylated by mitogenic signals, suggesting it could be activated during β-cell proliferation to drive requisite changes in metabolism. Our central hypothesis is that mitogens remodel β-cell lipid metabolism through Acly, and that this effect is necessary for optimal β-cell proliferation. Here, we will test how changes to islet lipid metabolism support β-cell proliferation in primary human islets using genetic or metabolic interventions coupled with targeted metabolomics and genomic assays. In Aim 1, we will monitor the effect of pro-proliferative drugs on islet lipid synthesis and test whether metabolites affected by mitogenic signals limit β-cell proliferation. To assess the requirement for lipid synthesis pathways downstream of Acly, we will determine how ACLY inactivation impacts human β-cell proliferation under the same conditions. In Aim 2, we will assess how the islet epigenome is remodeled in response to β-cell mitogens. Islets will be treated with pro-proliferative drug combinations, then we will perform ChIP-seq for histone modifications previously implicated in governing β-cell proliferation. We will also determine the epigenetic signaling function of Acly by testing its role in nutrient-stimulated histone acetylation in human islets. Understanding the metabolic pathways that support or augment pharmacological β-cell expansion could inform nutritional interventions to optimize β-cell regeneration by therapies currently in preclinical development.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Pulmonary hypertension (PH) is a cardiopulmonary disease that ultimately leads to right ventricular (RV) failure. Currently there are no approved therapies targeting the RV and most research is focused on reducing fibrosis, although it is unclear if this will ultimately improve RV pumping function. However, the orientation of collagen and cardiomyocyte fibers likely have a major influence on RV function and are largely overlooked in ongoing research and clinical practice. Furthermore, the role of the left ventricle (LV) in RV function is almost completely discounted, but previous research from the 90’s has suggested that the LV is more important for RV function than the contracting RV free wall. Our preliminary data shows that LV torsion rate is reduced in children with PH and in mice after pulmonary arterial banding (PAB), which is correlated with reduced RV ejection fraction in both species. However, when we induced LV pressure overload in the PAB mice (by partial aortic constriction), we improved their LV torsion rate and RV systolic function. This was further validated with in silico studies of the bi-ventricular heart, which showed that targeting LV torsion rate could improve RV systolic function, but it depends on RV free wall fiber orientation. Therefore, these results left us with the following questions: (1) What new orientation do the fibers adopt in response to pressure overload (our preliminary results are not consistent with others), and does this new orientation improve or worsen RV function? (2) How does RV fiber re-orientation impact LV-to-RV mechanical assistance during systole? (3) How does the transient fiber re-orientation and stiffening impact mechanical stress/strain within the tissue, and how does that impact -or is driven by- gene expression? In this study, we will combine the PAB mouse model with in silico simulations to investigate how changes in RV fiber orientation and stiffening, in the RV free wall, impact RV pumping function. Then, we will combine PAB with aortic constriction to study how RV remodeling interferes with LV torsion and if this interrupts LV-to-RV mechanical assistance during systole. Finally, by collecting a time course dataset of imaging and gene expression, we will identify genes that are directly impacted by changes in mechanical stress and expose how they trigger their downstream remodeling pathways. The questions being answered in this project will lead to a better understanding of how RV structural remodeling, in response to pressure overload, impacts RV function and interventricular coupling, and identify target genes governing this process for future studies.

NIH Research Projects · FY 2026 · 2023-05

Colorado has a long and storied history of heterogeneity-related ARDS, pneumonia, and sepsis (APS) research. After the initial Lancet description of the acute respiratory distress syndrome (ARDS), Colorado investigators subsequently identified pneumonia and sepsis as diagnoses associated with an increased susceptibility for ARDS. Since that time, Colorado investigators (including many key personnel on this proposal) have reported seminal discoveries regarding heterogeneity of ARDS, pneumonia, and sepsis including identification of a) alcohol use disorders (AUDs) as the first co-morbid conditions that increase susceptibility to ARDS, b) AUDs deleterious impact on sepsis mortality, c) and sex, racial, and ethnic differences in ARDS and sepsis epidemiology. Simultaneously, Colorado investigators have been unraveling the basic mechanisms of APS focusing on heterogeneity in the host inflammatory response. As a higher proportion of patients began to survive APS, we expanded our research to examine survivorship focusing on neuromuscular dysfunction. These studies have resulted in enhanced ways to diagnose weakness and improve our understanding of the neuromuscular trajectory of recovery in APS survivors. We propose to conduct two clinical center-specific scientific projects demonstrating the breadth and depth of our research that spans acute APS severity and its recovery trajectory in survivors. The acute project will identify distinct mononuclear cell endotypes present in the lungs and blood of APS participants with acute respiratory failure using bulk RNA seq. The recovery project will establish whether neuromuscular endotypes, based primarily on a single time-point nerve conduction study, can identify distinct and clinically relevant trajectories of recovery. We will also explore the cross-cutting theme of how AUDs impact APS heterogeneity. Building upon a strong and persistent research foundation, we have the research infrastructure to achieve all the outlined goals and are poised to be a strong contributor to the national APS consortium. As an original and integral member of the ARDS and then PETAL Network (for over 28 consecutive years), our multi-disciplinary and collaborative research group is experienced in conducting high quality NIH-funded prospective cohort research. In 2021, the Colorado research group enrolled 554 APS participants into clinical and translational research studies. This number far exceeds the APS consortium requirement of 240 APS patients per year. We also have extensive expertise academic rural recruiting historically underrepresented communities (Latinx, Black, and Indigenous) from both and community hospitals. For this proposal, we will also enroll participants from the often overlooked community, ensuring their representation in the APS consortium.In summary, the Colorado APS Center plans to exceed our enrollment obligations; maintain excellence in the quality of protocol compliance, data acquisition, and regulatory responsibilities; actively contribute to the steering committee and other APS committees; and most importantly advance science and contribute to improving the care of APS patients.

NIH Research Projects · FY 2024 · 2023-05

Project Summary The goal of this exploratory proposal is to deliver and express genetic material in the ferret model system early in development. Our strategy is based on an existing method developed in mouse for the rapid and spatially localized expression of DNA plasmids in postmitotic cells, a technique termed ‘iontoporation’. We aim to express CRISPR/Cas9 into ferret primary visual cortex (V1) to manipulate the expression of specific genes critical for the development and function of visual circuitry. Ferret is an ideal model system, as they are born relatively immature and visual circuit development is well-characterized. In this application, we propose to optimize and validate iontoporation in ferret to knockdown the expression of N-Methyl-D-aspartic acid receptors (NMDARs) to elucidate their functional role in the formation and function of V1 circuitry. We will validate the technique in vitro and in vivo, and start obtaining preliminary data for a collaborative R01, on the role of NMDARs in the development and function of local and long-range (between columns) horizontal connections in layers 2-3 of ferret V1. Successful development of this approach will open the door to genetic manipulation in a higher mammal with cortical columnar organization of the neocortex similar to primates and humans. It will also allow the development of genetic models of brain pathologies such as Schizophrenia.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Scientific Abstract of proposed research project Immune checkpoint blocker therapy has revolutionized our clinical approach in cancer therapy. However, the overall response rate still has room for improvement and varies greatly in different cancer types. The redundant but unique role of immune checkpoints in T cell immunity propels us to further study the role of novel immune checkpoints in cancer therapy. The BigLEN/GPR171 interaction is a newly identified GPCR pathway that has been reported to regulate food uptake and anxiety. Though GPR171 is commonly used as a T cell signature gene, its potential role in T cell immunity has not been explored. Our recent studies have implicated that the GPR171/BigLEN axis is a new T cell checkpoint pathway that can be modulated for cancer immunotherapy. We found that GPR171 is transcribed in T cells and its protein expression is induced upon antigen stimulation. The neuropeptide ligand BigLEN interacts with GPR171 to suppress T cell receptor- mediated signaling pathways and to inhibit T cell proliferation. Loss of GPR171 in T cells leads to hyperactivity to antigen stimulation and GPR171-deficient mice exhibit enhanced antitumor immunity. Blockade of GPR171 signaling by an antagonist promotes antitumor T cell immunity in various mouse tumor models. Our preliminary data further implicate that GPR171 can be an inhibitory receptor on T cell for cannabinoids. In the proposed study, we will dissect the role of GPR171 ligands in GPR171-mediated antitumor suppression. We will further determine the molecular mechanisms that GPR171 signaling inhibits anticancer T cell response. The approach of GPR171 blockade for cancer therapy will be tested in clinical tumor models, including chimeric antigen receptor T-cell therapy and humanized mouse model together with anti- PD-1 therapy. By the completion of these studies, our discovery that GPR171 is a receptor for cannabinoids will help to better understand the impact of cannabis usage in cancer treatment, more importantly, provide new strategies of cancer immunotherapy.