Case Western Reserve University

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY We propose to purchase a new Iconeus One functional ultrasound (fUS) neuroimaging system from the company, Iconeus, to enhance the biomedical ultrasound and neuroimaging capabilities of the Imaging Research Core of Case Western Reserve University (CWRU). The fUS system offers unique capabilities in the neuroimaging space, which are currently unmet by any other instrument. More specifically, fUS fills a critical gap between functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and standard small animal optical imaging methods by providing a method of acquiring ultra-fast (1 Mhz), high-resolution (<50 micron spatial resolution) data, which is highly sensitive to changes in cerebral blood volume and vascular morphology. The main points of distinction of the fUS system from fMRI are 1) the ability to acquire at an unprecedented temporal and spatial resolution changes of brain vasculature and cerebral blood flow in awake, mobile mice and rats; 2) a mobile, plug-and-play platform that can be relocated easily, if need be, to the site of an experiment; and 3) substantially lower cost and greater accessibility compared to an fMRI scanner. Moreover, it does not require the high maintenance costs of an MRI. Acquisition of this system would add value and advance numerous ongoing projects in brain imaging, neural engineering, deep brain stimulation, neurodegenerative diseases, pain, and development of theranostics for neuro-oncology applications. The fUS system would be the first of its kind in the greater Cleveland area and would enable investigators from CWRU and the surrounding four clinical affiliates (the Cleveland Clinic Foundation, University Hospitals Cleveland Medical Center, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and The MetroHealth System) to carry out cutting edge research in these very important fields. This new preclinical fUS capability will also be available to all regional investigators. A highly interdisciplinary group of 16 Major Users from all these institutions will work on collaborative projects using the Iconeus One. Ultimately, our goal in acquiring this advanced neuroimaging technology is to better visualize, design, evaluate, and translate treatments for neurological disorders. An advisory committee of highly experienced ultrasound and neuroimaging faculty at CWRU and external institutions will oversee the organization and usage of the system and recommend policies to maintain system performance and maximize utilization. These advancements will drive our research forward and ultimately improve human health.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY High-risk human papillomavirus (HR-HPV) is a known cause of >95% of cervical cancer and cervical intraepithelial neoplasia (CIN). Prophylactic vaccines are available against the most carcinogenic types and are highly effective, but the number of people receiving the vaccine is low, and vaccines are ineffective in women who have already developed CIN. Initial infection of HPV results in low-grade CIN (CIN1) and is mostly cleared by the immune system. However, failure of the immune system to clear HPV can lead to chronic infection and risk of high-grade CIN (CIN2 or CIN3) or invasive cervical cancer. There is emerging evidence that the vaginal microbiome plays an important role in this process however the mechanisms linking vaginal bacteria with host responses to HPV infection and cervical neoplasia progression are underexplored. Antibody binding to microbes can alter the structure and function of the microbiome, however this has not been comprehensively characterized in the female reproductive tract. Our overall hypothesis is that the proportion, abundance and/or functional profiles of antibody bound and unbound vaginal bacteria will differ between women with and without HPV infection, as well as those with normal cervical pathology, low-grade cervical lesions, and high-grade cervical lesions. Our overall goal is to define the associations between the proportion and abundance of antibody bound and unbound bacteria and their functions in these comparison groups. In this proposal we will utilize vaginal samples already collected from a comprehensive observational cohort (THRIVE HPV) that is specifically recruiting women with an abnormal pap smear test and following them longitudinally for 2-3 years to determine HPV status and regression or progression of cervical dysplasia. We will sort bacterial cells from vaginal swab samples at baseline enrollment into antibody bound and unbound fractions, then perform metagenomics to determine the abundance, species/genera, and functions of bacteria that are associated with antibody bound or unbound bacteria in each comparison group. A unique feature of this proposal is having access to longitudinal samples from a diverse (>50% African American) cohort for this and future studies to determine antibody bound and unbound bacterial stability over time, with comprehensive medical and pathological information. To the best of our knowledge, our work will be the first to define antibody bound and unbound vaginal bacterial populations at the species/strain level in women with or without HPV infection or different cervical pathologies. We anticipate this study will reveal antibody bound bacterial populations that are associated with a healthy or diseased microenvironment that can be identified as high-risk profiles needing to be monitored for treatment or targeted for novel immunological therapeutics.

NIH Research Projects · FY 2024 · 2024-08

The defining pathological hallmark of Lewy body diseases (LBDs) is the accumulation of misfolded a-synuclein (aSyn) aggregates, which are heavily phosphorylated at Ser129 (pS129-aSyn). LBDs comprise an array of often overlapping conditions, including Dementia with Lewy bodies (DLB), Parkinson’s disease (PD), Parkinson’s disease dementia (PDD), and PD with AD pathology (PD/AD). aSyn is known to regulate synaptic vesicle (SV) trafficking, including endocytosis. However, how aSyn regulates the endocytic process is poorly understood. In the pathological setting, aSyn pathology is transmitted from cell to cell through the secretion, internalization, seeding, and transport of misfolded and toxic aSyn. Despite much progress in understanding these processes, mechanisms governing the internalization of misfolded aSyn, an early step in its cell-to-cell transmission, remain unresolved. Based on preliminary studies, our overarching hypothesis is that the binding of b-arrestin2 (bArr2) to pS129-aSyn regulates membrane endocytosis and that the LMBRD2-bArr2 complex mediates the internalization, transmission, and toxicity of pathological aSyn. Utilizing cellular models and animal models combined with pathological, behavioral, electrophysiological, biochemical, immunohistochemical, in situ protein-protein interaction, and unbiased proteomics studies, we will validate, dissect, and elucidate the mechanistic basis of the bArr2-LMBRD2 pathway in the regulation of aSyn function, accumulation, transmission, and toxicity.

NIH Research Projects · FY 2026 · 2024-08

Project Summary Dysfunctions in mitochondria and mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) are associated with the accumulation of amyloid β (Aβ) and hyperphosphorylated tau in Alzheimer's disease (AD). Mitochondrial protein, CHCHD10, plays a pivotal role in governing various mitochondrial functions, including respiration, genome stability, dynamics, cristae organization, and oxidative phosphorylation. In our preliminary study, CHCHD10 declines in the brains of APP/PS1 mice and human AD patients, which negatively correlates with Ab levels. Restoration of CHCHD10 in APP/PS1 mice reduces AD pathogenesis in vivo. In transfected cells, wild-type CHCHD10 promotes mitochondrial respiration as well as mitophagy and autophagy via PINK1/Parkin and p62/LC3 pathways. In addition, increasing CHCHD10 mitigates MAM hyperactivity in AD. We have identified specific small DNA oligos (antagoNATs) derived from CHCHD10 natural antisense transcripts (NATs) that effectively augment CHCHD10 levels in both mouse and human cells. Our preliminary study shows that CHCHD10 antagoNATs increase endogenous CHCHD10 protein both in vivo in mice brains and in vitro in human cell lines. This study has two principal objectives: (1) to evaluate the therapeutic potential of CHCHD10 antagoNATs by assessing their protective effects on these phenotypes in APP/PS1 mice and human neurons, and (2) to delineate the intricate interplay between CHCHD10, MAM, and mitophagy, thus elucidating their collective role in ameliorating AD pathogenesis. Successful conclusion of this study will: (1) facilitate the CHCHD10 antagoNATs as a therapeutic strategy for AD; (2) provide a mechanistic basis and insights into CHCHD10-associated mechanisms in AD.

NIH Research Projects · FY 2024 · 2024-08

Project Abstract This project aims to better understand the molecular mechanisms of HIV spread through cell-cell contact across a virological synapse (VS), a specialized structure formed between HIV infected cells and target cells. We have developed a novel flow cytometry assay for quantifying and purifying infected cell-target cell pairs. We have generated infected producer cells that stably express ascorbate peroxidase (APEX) enzymes localized to different subcellular compartments. Upon addition of hydrogen peroxide and biotin-phenol, APEX generates short-lived BP radicals that covalently biotinylate proteins and RNAs within a few nanometers of the enzyme. We will use APEX localized to nucleus, cytoplasm, ER, plasma membrane, and mitochrondria to provide a robust analysis of how host and viral proteins and RNAs are regulated by VS formation. The successful completion of this grant will identify determinants of the VS formation and its composition, identify changes to host cell proteins following VS formation with an emphasis on RNA binding proteins, and determine how host and viral RNAs are regulated by cell-cell contact. Aim 1: Identify determinants and composition of virological synapse (VS) formation. Aim 2: Determine how VS formation affects protein abundance and localization within infected cells, with an emphasis on cellular RNA binding proteins. Aim 3: Determine how cell-cell signaling during VS formation impacts the RNA regulatory landscape of infected cells.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract: The anaphase-promoting complex/Cyclosome (APC/C) is a well-defined multi-subunit E3 ubiquitin ligase that regulates targeted cell cycle regulators for degradation by the Ubiquitin Proteasome Pathway (UPP), promoting cell cycle progression from metaphase to anaphase and being involved in G1 phase maintenance. The APC/C E3 ligase complex is evolutionarily conserved and relies on two adaptor proteins, Cdc20 and Cdh1, to recognize different target proteins and regulate cell cycle progression. However, compared to Cdc20 that is subjected to Cdh1-mediated destruction, regulation of the E3 ligase activity of Cdh1 is not well known yet. Previous study has shown that there were 19 serine and threonine residues on Cdh1 that can be phosphorylated by multi-kinases in vivo, indicating that the phosphoregulation of Cdh1 is much more complex. In the present proposal, I found that CDK4 can phosphorylates Cdh1 in vitro and modulates its E3 ligase activity. Furthermore, we found that the phosphorylation of Cdh1 by CDK4 can be recognized by the Pin1 proline isomerase, facilitating Cdh1-Pin1 complex formation. In keeping with this notion, employment of the CDK4/6 inhibitor or mutating the phosphorylation sites can disrupt the Cdh1-Pin1 interaction. Consequently, Cdh1 can mediate Pin1 for polyubiquitination and degradation. As such, depletion of endogenous Cdh1 abolished the Pin1 inhibitor treatment induced Pin1 degradation in cells. Importantly, the Pin1 inhibitor-induced cell proliferation suppression was also abolished in Cdh1-null MEFs, suggesting the functional presence of Cdh1 is required for Pin1 inhibitor-induced cell proliferation suppression. In addition, combination of the CDK4/6 inhibitor and Pin1 inhibitor exhibits significantly enhanced suppressing effect in breast cancer cells. In the first Aim of this proposal, I am going to explore the role of CDK4 kinase in regulating the E3 ligase activity of Cdh1 (Aim #1). Therefore, the second Aim in this proposal will be exploring the potential role of Cdh1 in mediating Pin1 inhibitor treatment induced Pin1 protein destruction (Aim 2). Together, these results implicate a functional role of the CDK4/Cdh1/Pin1 signaling axis in regulating cell proliferation, and provide rational for combining the CDK4/6 inhibitor and Pin1 inhibitor to treat breast cancer.

NIH Research Projects · FY 2025 · 2024-08

Project Summary The Triple Risk Model describes SIDS occurrences when an intrinsically vulnerable infant experiences an exogenous insult resulting in a chronically hypoxic/hypercapnic environment particularly during a critical developmental period. SIDS pathophysiology includes evidence of chronic hypoxia exposure, brainstem gliosis and serotonergic abnormalities, as well as respiratory/autonomic dysfunction and carotid body abnormalities. Although animal models have been instrumental in advancing our understanding of SIDS, the lack of models that recapitulate the hallmark features has hindered our ability to confirm SIDS pathophysiology and resolve the major hypothetical/proposed features. We resolved this hurdle after discovering a rat model that closely simulates postnatal hypoxia component of SIDS as an exogenous stressor in a vulnerable neonate. In this model, prolonged/sustained (days) hypoxia exposure during a uniquely critical period of postnatal development results in spontaneous unexplained death several days later. Importantly, in both published and preliminary studies (this proposal) the model recapitulates ALL of the aforementioned SIDS features. Here, using our novel model, we propose the novel hypothesis that the hallmark brainstem abnormalities (microglia and 5-HT) in SIDS is in response to chronic disruption of carotid body afferent inputs into the brainstem. We propose that these disrupted inputs over several days are sufficient to elicit a localized brainstem microglial response (as seen in SIDS), ultimately leading to the fatal abnormalities in brainstem neurochemistry in key respiratory/autonomic control regions. A particularly novel component of our proposal is the discovery of a microglial inhibitor, which prevents the adverse effects of hypoxia exposure, thus for the first time in any setting, we may be on the path to a preventative measure against SIDS. We also propose that aberrant expression of several components of the extracellular matrix may be a new central (brainstem) and peripheral (carotid body) pathophysiological mechanism in SIDS. Finally, given the compelling similarities in our model with SIDS cases, we are poised to assess serum and urine biomarkers for identifiers of at-risk infants and predictors of later mortality. Overall, this proposal will: 1) be fundamental to our understanding of respiratory and autonomic dysfunction associated with SIDS, 2) provide a mechanistic perspective on the root cause of the common brainstem abnormalities, 3) discover potentially new SIDS pathophysiology (per the requirements of the NOSI, and 4) reveal a glimmer of hope at a prophylactic treatment toward SIDS prevention.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT This collaborative project integrates fundamental molecular biology, cell level biophysics, animal-level physiology, and computer modeling to advance understanding of molecular mechanisms by which inherited mutations in cardiac myosin binding protein C (cMyBP-C) cause disease. Some individuals who inherit mutations in this protein are at increased risk of developing hypertrophic cardiomyopathy but clinicians know that not all mutations lead to significant disease. Linking genotype to phenotype is particularly challenging for missense mutations as these often cause cMyBP-C molecules with abnormal function to be expressed in a patient’s heart. More than 1000 missense mutations have already been identified but there is rarely enough clinical information to determine the severity and/or best treatments for a given variant. Therefore, most are still characterized as variants of unknown significance. Further, the field’s understanding of the basic mechanisms by which missense mutations in cMyBP-C cause disease is limited because cMyBP-C exhibits complex behaviors and it’s N-terminal and central domains can impact contractile function in diverse ways by interacting with both myosin and actin. While it seems likely that the mutation’s location on the molecule determines its impact on contractile function, mechanistic analyses of the region-specific molecular underpinnings of cMyBP-C missense variants has not yet been performed. The large number of variants makes it impractical to create animal or cell-based models for each missense mutation. This project advances the field by combining strategically selected biological experiments with computer modeling to develop a data-driven pipeline that can ultimately be used to identify which missense mutations currently classified as variants of unknown significance pose the greatest risk to patients and the best way to treat each variant. To address this important problem we assembled a multidisciplinary research that will integrate experimental approaches that span temporal and spatial scales, and complementary expertise in basic mechanisms of cMyBP-C and clinical presentation of cMyBP-C related HCM. The research plan has 3 Aims: 1) Predict the mechanisms and severity of missense mutations in cMyBP-C that cause hypertrophic cardiomyopathy. 2) Test predictions of cardiac phenotype using AAV9 to express mutant cMyBP-C in mouse hearts. 3) Use a data-driven mechanistic approach to determine the most effective treatment for cMyBP-C variants. The plan is highly innovative and makes intelligent use of the skills and resources of four leading investigators. The team are committed to developing shared resources and will publish their computer code as open-source projects as well as sharing their cell and animal-level data as freely-accessible databases to accelerate future research.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Across the animal kingdom, specialized neurosecretory cells coordinate endocrine and central nervous system signals to construct neural circuits and maintain homeostasis. Insulin/insulin-like growth factors (IGFs) are key secreted neuropeptides whose signaling activities are strictly regulated by IGF-binding proteins (IGFBPs). Despite misregulated insulin/IGF signaling (IIS) being linked to the pathogenesis of neurodevelopmental disorders, the cellular and molecular mechanisms by which IGFBPs precisely tune IGF activity in the brain to sculpt neural circuits and modulate behavioral outputs are largely undefined. The Drosophila system provides an appealing model to address these questions given only two IGFBPs are encoded in the fly genome, Imp-L2 and Crimpy. Our lab discovered Crimpy and defined its role in facilitating TGFβ signaling at the peripheral neuromuscular junction, but its roles in the central brain and IGF regulation are completely unexplored. Our preliminary data suggest that Crimpy is required for axon morphogenesis and circuit formation of the mushroom body, the fly learning and memory center. We also demonstrated that Crimpy is required to promote wild-type sleep behavior. Notably, Crimpy’s cellular requirements for axon morphogenesis and sleep regulation were mapped to a cluster of 14 neurosecretory cells at the brain midline, termed insulin producing cells (IPCs), which produce and secrete insulin-like peptides/IGFs. Based on these results, we hypothesize that Crimpy tunes IGF signaling from IPCs to promote proper central brain circuit development and function. Through the use of innovative genetic schemes, immunocytochemistry, as well as behavioral and electrophysiological analyses, this proposal seeks to: 1) establish roles for Crimpy in IPCs for central brain morphogenesis and circuit formation, 2) assess Crimpy’s function in IPCs to promote wakefulness, and 3) define Crimpy’s regulatory actions on IGF activity/IIS. These studies will provide novel mechanistic insight into the regulatory mechanisms governing IGF activity to fill fundamental gaps in our understanding of IIS in constructing neural circuits and modulating behavior. In addition, these proposed goals and approaches will provide exceptional training opportunities to strengthen my technical and professional skills, and prepare me for independence as I advance in my scientific career.

NIH Research Projects · FY 2025 · 2024-08

This proposal seeks to develop new statistical methods applicable to studies of bundled interventions. Randomized clinical trials, including in dental research, often focus on multi-component and other complex interventions. Using such ‘bundled’ interventions is appealing as a way to increase power – as well as simplicity - of the study. An apparent disadvantage is that it is not clear how to assess the effects of individual components of bundled interventions, which of also of frequent interest. While the measurement of treatment compliance, and use of causal mediation analysis is commonly recognized as a possible approach, rigorous methods to identify and estimate causal effects of components are not available. The present research seeks to fill this important gap. We will first elucidate the assumptions under which causal mediation/path analysis can be used to determine causal effects of individual intervention components. We propose, as a novel and relevant estimand, what we refer to as a cluster-specific interventional effect. We will develop an extended mediation formula/simulation approach to estimating these causal effects. In our second aim, we will extend methods to handle repeatedly measured mediators and outcomes. As a novel aspect of this aim, new methods will be developed to analyze summary (or cumulative) measures in a way that respects the causal order of model variables. We will perform simulation studies to evaluate the properties of the new methods, and compare them to possible alternative approaches. In addition, we will develop sensitivity analysis methods to examine the impact of violations of model assumptions, including extended sequential ignorability as well as structural (e.g., no direct effect) assumptions. In particular, we extend a copula model approach, previously developed for the single mediator case, to perform sensitivity analyses in the context of more complex path models. We will develop an R package to allow user-friendly implementation of the new methods. The new methods will be applied to data from a recently completed cluster-randomized clinical trial of a multi-component intervention (including multiple provider-level components) to improve dental care utilization among 3 to 6 year old Medicaid-enrolled children attending well-child visits in primary care settings. Our analysis will assess the causal effects of individual components of this bundled intervention.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Glioblastoma (GBM) is a CNS tumor derived from glial cells. It is the most common and fatal primary malignant brain tumor in adults with a median survival of 15 months. Current treatment strategies involve surgical resection followed by radiotherapy with concurrent chemotherapy. Unfortunately, GBM is highly resistant to therapy and will inevitably recur, resulting in poor patient outcomes. GBM has been described as having an oxygen-deprived niche and a perivascular, oxygen-rich, niche. Deeper characterization of these tumor environments has revealed the presence of cellular heterogeneity, angiogenesis, and invasion in both niches. The critical cellular player involved with these tumor behaviors is the glioblastoma stem-like cell (GSC). GSCs seem to reside in both hypoxic (low oxygen level) and normoxic (normal oxygen level) niches yet maintain their stemness and self- renewal capacities across both conditions. Strategies to target these GSCs have proven to be unsuccessful so far but remain a promising avenue for research. There is a need to develop a greater understanding of GSC biology, starting with the regulatory mechanisms with which they adapt to the hypoxic environment of GBM and promote tumor growth. However, most studies exploring GSCs are performed in normoxic conditions. Further, the central aspect of post-transcriptional gene regulation, translation, where genetic information is converted to functional proteins, remains relatively unexplored. The objective of the proposed research is to study the translation (mRNA-to-protein) regulatory step of protein synthesis in GSCs, as opposed to the more commonly explore transcription (DNA-to-mRNA) regulatory step, with the desired outcome of illuminating systemic programs for hypoxic adaptation. To accomplish this objective, our approach in Aim 1 will use ribosome profiling (RIBO-seq) in conjunction with RNA sequencing (RNA-seq) to offer a quantitative understanding of transcriptional and translational regulatory pressures on mRNA transcripts. I will utilize both techniques to clarify the influence on hypoxia on translational gene regulation in GSCs. I expect to find genes and gene-sets, representing biological pathways, that are modulated by hypoxia predominantly through translational regulation, revealing a novel list of candidates for future therapy. To validate this discovery framework, I will perform an array of functional studies on selected genes. In parallel, Aim 2 will focus on a particular set of translational machinery known as the Integrated Stress Response (ISR), whose members sense cellular stress and modulate translation of survival-associated transcripts. With burgeoning evidence of aberrant ISR behavior in malignant cells, interrogating the mechanism through which the ISR may mediate translational changes in hypoxic GSCs will present another avenue through which to halt such adaptive behaviors. Altogether, this proposal will promote further understanding of GSC biology, with respect to translation regulation, thereby unveiling novel targets for future GBM therapy and ultimately contribute to brain tumor patient care.

NIH Research Projects · FY 2024 · 2024-07

Project Summary It is estimated that up to 0.6% of the US population is transgender, which describes someone whose gender identity is incompatible with their sex assigned at birth. Gender affirming medical care can include vaginoplasty, which is the surgical creation of a vulva and neovagina. Transgender women have increased odds of testing positive for sexually transmitted infections (STIs) including human immunodeficiency virus (HIV) compared to cisgender women, which is maintained after adjusting for high-risk behaviours, suggesting biological factors unique to the neovaginal microenvironment may contribute to this increased risk. Despite this, little is known about the neovaginal microenvironment, which could have important implications for both post- operative care and long-term mucosal health of people with neovaginas. The neovaginal microbiome is not well-defined, but typically has greater microbial diversity compared to the natal vaginal microbiome, and contains species that are more typically detected in the penile, intestinal and skin microbiomes. In cisgender women vaginal microbial dysbiosis has been linked to increased genital inflammation, gynecological symptoms, and an increased risk of acquisition of STIs including HIV, but it is unknown if the neovaginal microbiome has a similar impact on gynecological health. A major gap in knowledge is defining the optimal neovaginal microbiome, including an understanding of the colonization and stability, and relationship to gynecological health, which is vital for care of people with neovaginas. Indeed, current post-operative and long-term care recommendations differ greatly between surgical centers. In this proposal we will define the neovaginal microbiome in transgender women who have undergone (“post-op”) or are scheduled to undergo (“pre-op”) vaginoplasty, and investigate associations of the microbiome to genital inflammation and clinical outcomes including gynecological symptoms. This study will be the first to comprehensively profile the neovaginal microenvironment, which is critical to provide evidence-based care guidelines to people with neovaginas.

NIH Research Projects · FY 2024 · 2024-07

Project Summary/Abstract Transfer RNA (tRNA) biogenesis is essential for determining the pool of tRNAs that set the translational state of cells, and defects in the tRNA biogenesis machinery are associated with several types of neurodegenerative diseases. While bulk tRNA-sequencing assays of tissues and cell cultures have revealed that tRNA expression and modification are regulated in tissue and disease-specific manners, technology to date has hindered our ability to define tRNA processing, expression, or modification changes in neurodevelopment and neurodegeneration at the level of single cells, limiting important insight and therapeutic development. In this application, we propose to develop and apply a novel tRNA sequencing method to stem cell derived neurons and cerebral organoids to reveal complex tRNA processing and modification patterns at the single cell level for the first time. We will utilize preexisting laboratory models to establish and optimize our technology (Aim 1) before profiling cerebral organoids with pathogenic variants in CLP1 and isogenic controls (Aim 2). These findings will reveal the complexity of tRNA processing and modification at unprecedented cellular level resolution. In addition to neurodevelopment and neurodegeneration, our research will have broad impacts on the study of tRNAs across all tissues, as it will be applicable to any frozen or fresh tissue source. Outside of neurobiology, it will also benefit other fields such as cancer biology and virology where tRNA modulation is known to be important.

NIH Research Projects · FY 2025 · 2024-07

Summary Congenital defects affecting the formation of the skull roof, such as craniosynostosis or persistent fontanelles, occur as a result of abnormal calvarial growth and differentiation. We lack a basic understanding of how calvarial bones grow, which in turn impacts the position, patterning, and fusion of sutures, and thus etiology of these disorders. The Harris and Atit laboratories have uncovered an unexpected and intriguing role for cellular sensing of graded fibronectin matrix in preferentially regulating apical expansion of calvarial progenitors during mouse development. When cellular lamellipodia are inhibited, mouse calvarial osteoblasts fail to appropriately migrate resembling defects seen when we conditionally delete fibronectin. These findings are bolstered by data that fibronectin is misregulated in patients with craniosynostosis as well as animal models of this disease. We propose that graded fibronectin may act as a substrate for coordinated migration of calvarial osteoblast progenitors over the skull roof. Our central hypothesis is that calvarial growth and suture patency are dependent on fibronectin-directed calvarial progenitor cell expansion. Through three focused mechanistic and translational aims, we will directly test this model and hypothesis of fibronectin substrate-mediated migration underlying a diverse number of cranial pathologies. First, we will assess outcomes of altered fibronectin expression in regulation of calvarial growth. Second, using newly established genetic lines in mouse and zebrafish, we will test the dependence on fibronectin adhesion and the role of lamellipodia-dependent cellular sensing of an extracellular gradient in apical expansion of calvaria. Third, we will capitalize on diverse mouse models of craniosynostosis to assess commonality of fibronectin disruption in clinically relevant dysmorphologies and whether decreasing fibronectin expression rescues craniosynostosis in in vivo. Our unique genetic tools in both mouse and zebrafish will allow us to define the function of fibronectin-guided, lamellipodia-based, collective cell movement in vivo during calvarial bone expansion and the impact of fibronectin deficiency on suture patency. Results from these studies will help detail substrate-mediated cell migration of osteoblast progenitors and will lead to new strategies for targeted therapies of calvarial bone defects and craniofacial disorders.

NIH Research Projects · FY 2026 · 2024-07

Project Summary Incidence of HPV+ OPC has been increasing for several decades and this upward trend is expected to continue until at least 2060. Current first-line modalities to manage HPV+ OPC patients are effective but not without limitations. A key event in HPV-driven tumorigenesis is the inactivation of the p53 tumor suppressor program. HPV oncogene, E6, inactivates p53 through two distinct pathways: HPVE6 promotes the assembly of the HPVE6-E6AP-p53 trimeric protein complex resulting in ubiquitination and degradation of p53, and HPVE6 directly binds to p300 to block p300-directed acetylation and activation of p53. We hypothesized that disrupting the HPVE6-p300 interaction will liberate sufficient p300 to restore p53 and p300 functionality simultaneously in HPV+ OPC. Our team initiated a drug discovery platform to target the HPVE6-p300 interaction. Our work showed that in HPV+ OPC models: (a) HPVE6 binds to the CH1 domain of p300, (b) our lead molecule, OHM1, a CH1/p300 ligand, disrupts HPVE6-p300 interaction and reactivates p53 and p300, (c) OHM1 is active in vitro and in vivo, and (d) concurrent OHM1+cisplatin combination treatment yields durable complete anti-tumor responses in vivo. Our results are very compelling and, supports further research and development of dual p53 and p300 reactivation as a therapeutic strategy for HPV+ OPC. In this project, we propose to extensively determine the mechanisms of action of p53 and p300 reactivation in response to OHM1 in HPV+ OPC. The specific aims are: (1) determine if OHM1 modulates the p53 post-translational modification code to control p53 functionality and levels, and promote anti-cancer activity in HPV+ OPC, (2) to determine if OHM1 reshapes the tumor-microenvironment and boost immunotherapy response in HPV+ OPC.

NIH Research Projects · FY 2025 · 2024-07

Project Summary With cells constantly encountering DNA damaging agents, mechanisms for prompt DNA repair are crucial for maintaining genomic stability. Aberrations and mutations in the proteins involved these processes are closely associated with a plethora of diseases, including cancer and neurological disorders. Among various types of DNA damage, single-strand breaks (SSBs) are the most prevalent, occurring at a rate of 55,000 per cell daily. Understanding the repair of SSBs within chromatin through the base excision repair (BER) mechanism is the primary focus of the proposed research project. The proposed research project primarily aims to unravel the complexities of SSB repair within chromatin through the BER mechanism. This multifaceted pathway involves poly-ADP-ribosylation (PARylation) and histone acetylation, both closely associated with DNA repair. PARP1, the most abundant member of the PARP family, is the enzyme that confers poly-ADP-ribosyl groups to histones upon detection of SSBs. This PARylation then recruits and activates various repair proteins like DNA Ligase III (LIG3) and Polynucleotide Kinase 3'-Phosphatase (PNKP). PNKP has dual functions in the process, particularly for SSBs induced by radioactivity, by adding a phosphate group to the 5'-end of DNA breaks and removing one from the 3'-end. This prepares the DNA ends for the action of DNA ligases, such as LIG3, thereby playing a vital role in DNA repair mechanisms. This proposal aims to understand how three DNA repair proteins—PARP1, LIG3, and PNKP—recognize and repair SSBs within the context of nucleosomes and to explore the interplay between PARylation and acetylation of histones in these processes. We will investigate how PARP1 identifies and recognizes SSBs within nucleosomal contexts, a critical phase in the DNA damage repair process. Additionally, we will explore the roles of enzymes LIG3 and PNKP in repairing SSBs within nucleosomal structures. To achieve these goals, we will map the accessibility of SSB sites within nucleosomes using DNA libraries and next-generation sequencing, aiming to optimize the positioning of an SSB for facilitating the formation of nucleosome complexes with the DNA repair proteins. Subsequently, we will employ biophysical tools and functional assays to characterize these interactions and functions, both in the presence and absence of histone modifications. Finally, we will leverage state-of-the-art NMR techniques and cryo-EM to obtain detailed structural and dynamic information about the nucleosome when complexed with PARP1 (or its zinc finger domains), LIG3, and PNKP. The long-term goal of this research is to provide an enhanced fundamental understanding of BER mechanisms within nucleosomal contexts, laying a molecular foundation for the design of drugs and therapeutics that can beneficially modulate these mechanisms in various disease states.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY/ABSTRACT All genetic information is stored in DNA that is intricately wrapped by proteins to form chromosomes. Telomeres are the nucleoprotein complexes that cap and protect the ends of chromosomes to prevent them from fraying, fusing together, and degrading. In addition to capping and protecting the ends of chromosomes, telomeres regulate the recruitment of telomerase, a specialized enzyme that synthesizes telomere DNA to collaborate with replicative polymerases and ensure complete chromosome replication. Over the past several years, multiple single nucleotide polymorphisms have been identified in the genes of telomere end-binding proteins in patients diagnosed with a range of disorders, including many different types of cancer. These observations indicate that subtle changes in the structure and/or function of telomere proteins contributes to genome instability. POT1 (Protection of Telomeres 1) is the most mutated telomere protein associated with human disorders. POT1 forms a heterodimeric complex with another telomere end-binding protein, TPP1, to perform diverse but equally critical functions. Specifically, POT1-TPP1 binds to the extreme 3’ end of telomeres and helps to resolve DNA secondary structure, to recruit telomerase to the telomere, and regulate telomerase- mediated telomere synthesis. In addition, the POT1-TPP1 proteins shield telomere DNA from being recognized and repaired by DNA damage machinery. Finally, the POT1-TPP1 heterodimer exhibits extraordinary sequence specificity that provides discrimination against binding to RNA or to DNA with non-telomere sequence. The objective of the present proposal is to investigate the multiple and diverse roles of POT1-TPP1 in telomere maintenance. We will further interrogate the intricate details of telomerase-mediated extension of telomere DNA and identify how changes in nucleotide pools affect telomerase function and fidelity. Additionally, we will examine the role of FDA-approved and developing nucleotide and nucleoside analogs in telomerase rates and fidelity. To accomplish these objectives, we combine structure-function studies to determine the molecular interactions that dictate POT1-TPP1 function and we corroborate the mechanistic studies with cellular outcome. The results from this investigation will reveal both structural and functional alterations introduced by pathogenic mutations and drug administration and will be used to better understand the diverse functions of specialized telomere proteins and telomerase in maintaining telomere integrity and genomic stability. The project includes a translational component as we will define the understudied contributions that chemotherapeutic and antiviral agents have on telomere integrity. On a fundamental level, the work performed in this study will shed light on the assembly, organization, and functional motions that regulate chromosome end protection.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Nuclear receptors are steroid-dependent transcription factors that confer cell identity and function. They guide development, and maintain homeostasis in adult tissue, such as the testosterone responsive Androgen Receptor which governs male-specific organs and phenotypes. Aberrant AR-driven gene expression programs give rise to prostate cancer. When resistance arises after androgen-deprivation therapy (ADT) the disease advances to the much more lethal metastatic castration-resistant prostate cancer (mCRPC), often becoming resistant to ADT by overexpressing AR. New evidence suggests that many other transcription factors and epigenetic co-activator proteins may create disease-specific enhancers with the AR to drive tumors, especially in treatment-relapsed mCRPC. By harnessing sophisticated molecular biology and computational biology techniques, we will map the epigenetic mechanisms at work in mCRPC, in clinically relevant PDX models and cell lines with epigenomes closely matching mCRPC tumors. We will show how to stop the AR-axis using small molecules (AR in verse agonists, ARIAs) we designed, synthesized and developed with novel mechanisms of action, where the AR undergoes a chemically induced functional switch, causing the AR to suppress the tumor- driving genes it normally activates. We will study the way AR creates super clusters in the diseased genome, and how our new therapy works by altering the co-regulators recruited to AR-super clusters. At the conclusion of this work, we should have mechanistic understanding the AR under the influence of an inverse agonist, provide functional 3D epigenetic map of regulatory addictions in advanced PCa, and have explored therapeutic potential of promising new AR therapeutics.

NIH Research Projects · FY 2026 · 2024-07

Title: Assessment of the Impact of antihypertensive Medications on vascular and renal outcomes in Chronic Kidney Disease (AIM-CKD) Abstract Chronic kidney disease (CKD), characterized by enduring damage to kidney function, poses a significant public health concern, impacting around 15% of the adult population in the United States. CKD patients commonly experience heightened adverse cardiovascular effects, which are associated with an elevated risk of mortality. Foundational treatment strategies for slowing CKD progression involve blood pressure-lowering agents, including renin-angiotensin system inhibitors and calcium channel blockers. These medications also exhibit potential vasodilatory and anti-inflammatory properties. Nevertheless, the impact of these therapies on cardiovascular disease outcomes in CKD patients is still in debate, and the tradeoff between risks and benefits remains unclear due to limited clinical trials conducted thus far. To address this knowledge gap, we intend to leverage data from two extensive studies: the Chronic Renal Insufficiency Cohort (CRIC) and the Systolic Blood Pressure Intervention Trial (SPRINT). Our goal is to estimate and compare the effects of medications on cardiovascular risks in CKD patients using novel and advanced causal inference methods. We recognize that analyzing real-world data poses statistical challenges (e.g., time-varying treatment administration, competing risk of death, treatment combination). However, these complexities cannot be directly addressed by current causal inference techniques or may be addressed inefficiently. Our proposal focuses on time-to-event outcomes, which accommodate detailed information, including the timing and duration of disease outcomes that are crucial for understanding the natural progression of the disease and assessing the potential effectiveness of interventions. Preliminary work has demonstrated the promise of our novel dynamic propensity trajectory matching (DPTM) techniques, which guarantee further evaluation through theoretical and extensive simulation studies conducted under various scenarios. Ultimately, we aim to develop user-friendly R software packages and a Shiny app to facilitate the broader use of our research findings for the benefit of the public.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY RNA binding proteins (RBPs) play critical roles in all aspects of RNA metabolism, with significant implications in physiological and pathological settings. A crucial factor in these processes is the exact location of these RBP:RNA interactions within the cell. These interactions are pivotal when cells adapt to stress and are often dysregulated in disease. Our current tools fall short in identifying RBP targets with subcellular resolution. This proposal introduces a groundbreaking approach to bridge this knowledge gap. We aim to develop and apply innovative tools designed to capture subcellular information for individual RBP:RNA binding events at a systems scale within living cells. Using cellular models that mimic halted protein synthesis, we aim to build a comprehensive spatial map of RBP:RNA interactions, shedding light on the remodeling of these networks during changes in post-transcriptional and translational control. At the level of individual RBPs, we will explore the temporal sequence of events by which specific RBPs regulate the localization, stability, and translation of their target RNAs, especially during stress-induced relocalizations. Using cutting-edge proximity labeling and isolation techniques, we will map RBP targets across subcellular space and integrate them within the broader landscape of cellular RNA networks and interactions. These data will be analyzed using state-of-the-art computational approaches and integrated with other high-throughput datasets such as Riboseq and RNAseq. This integrative analysis will enable us to construct and iteratively test predictive models of RBP activity based on their spatial distribution. Building off of this, we will apply these innovative methods to discern RBP:RNA regulatory programs in difficult to isolate membrane-less organelles, using static and oscillating mammalian stress granules (SGs) as our investigative platform. Overall, this proposal lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions. Our comprehensive approach, bridging experimental and computational analyses, promises to uncover novel mechanisms of RBP action during changing cell states, providing insights that may guide future therapeutic strategies against an array of RBP-associated diseases that impact human health.

NIH Research Projects · FY 2026 · 2024-07

Abstract. Prostate cancer (PCa) is the most prevalent cancer among men in the United States and radical prostatectomy remains one of the three treatment options for these patients. Screening with serum prostate specific antigen (PSA) allows for 78% of prostate cancers to be diagnosed at the early localized stage, facilitating therapy with radical prostatectomy (RP) or radiation therapy. However, ∼ 20% patients present with high-risk PCa, i.e., positive surgical margins (PSM) discovered after surgery during pathology of resected tissues. These patients with high-grade tumors also have a high risk of biochemical recurrence (>60%) and will ultimately develop lethal metastatic disease. Surgical approaches to prostate cancer are also associated with significant morbidity, e.g. incontinence (3-74%) and impotence (30-90%) due to the close proximity of the prostate gland to critical nerves and muscles. Therefore, there remains an unmet clinical need to improve surgical techniques for identifying and removing all cancerous tissue without damaging surrounding tissues during prostatectomy and to prevent relapse. The goal of this study is to provide a means to address both unmet needs, i.e., to more completely remove cancers and at the same time generate an immune response to help prevent local recurrence and metastatic disease. In the proposed study we will develop a precisely targeted theranostic agent, PSMA-1-Pc413, for both fluorescence guided surgery and photodynamic therapy (PDT) to effectively treat prostate cancer. The overall approach is to use the highly expressed and tumor selective prostate specific membrane antigen (PSMA) biomarker to target a highly fluorescent and potent photosensitizer, Pc4, to prostate cancer tumors. Following IV injection and recognition of the PSMA receptor on prostate cancer cells, PSMA-1-Pc413 will be used for fluorescence image guided surgery to remove the PCa from the flank of mice. Following excision, the surgical field will be irradiated with light to stimulate PDT to remove any non-visible or non- resectable cancerous cells. Furthermore, PDT may stimulate a local and even systemic immune response limiting local recurrence and metastatic disease. We will first test our hypothesis in immune competent syngeneic mouse prostate cancer models. Since efficacy trials in mice are not always predictive of human results, we will also test the approach in an orthotopic canine prostate cancer model; dog pathology and physiology of prostate cancer is very similar to humans and dogs are often used in drug development trials. After probe injection, PDT will be performed and the canine prostate gland and any extra-glandular cancer tissues will be resected. Correlation of the probe to cancerous tissues and the impact of PDT on cancer and surrounding normal tissues will be assessed using histopathology. Targeting and PDT studies in dogs will substantially encourage the potential to use this agent in dogs with spontaneous forming prostate cancers (beyond the scope of these studies) and clinical translation of the developed agent. Our innovative, combined theranostic approach, if successful, will significantly alter the way prostatectomies are performed in the future by: 1)increasing surgical efficacy; 2) prolonging progression-free survival; and 3) decreasing morbidity. No such approach has been reported for PCa.

NIH Research Projects · FY 2025 · 2024-07

Abstract The most common malignancy for men in the western world is Prostate cancer (PCa), with a predicted 288,300 new cases and 34,700 specific deaths in 2023. With recent developments in comprehensive screening and biopsy strategies, the ten-year survival rate of PCa is largely improved to 98% and the disease specific mortality is reduced to only 4-8%. However, the widespread adoption of these strategies has also led to significant overtreatment of the disease, leading to a significant loss in patient quality of life with an added financial burden of 22 billion dollars annually in the U.S. alone. To better detect and guide biopsies, non- invasive magnetic resonance imaging (MRI) has emerged as an informative, accompanying tool along with the Prostate Imaging Reporting and Data System (PI-RADS), an internationally established scoring system for characterizing the risk of clinically significant PCa (csPCa) in focal lesions detected on MRI. While aiming to defer biopsies for low-risk patients whenever possible, ~30% of patients with a negative MRI still end up proceeding to biopsy due to suboptimal negative predictive values with current MRI techniques (~90%). This leads to unnecessary biopsies and post-procedure complications for a population in which the prevalence of csPCa is only around 8%. The majority of detected PCa (70%) is localized in the peripheral zone of the prostate. Thus, there is an urgent need for novel, non-invasive imaging techniques to improve our capability to more definitively rule out csPCa in the peripheral zone of the prostate to avoid unnecessary biopsies, complications, and costs. Our team has pioneered the prostate Magnetic Resonance Fingerprinting (MRF) technique, which simultaneously quantifies T1 and T2 in ~40 sec per slice. We propose to develop a rapid and reproducible MRF method to quantitatively and more accurately characterize prostatic peripheral zone tissue in order to limit overdiagnosis and overtreatment for patients with no csPCa. We will develop novel prostate MRF techniques to provide simultaneous and motion-robust T1, T2, and diffusion quantification (Aim 1). Rapid and whole-gland imaging will be achieved by leveraging novel deep learning techniques and multi-slice imaging. Deep-learning-based prostate segmentation derived from MRF signal evolutions will be further developed to automatically extract quantitative metrics from the peripheral zone for post-processing (Aim 2). These developed methods will be applied in a diagnostic study of a population with clinical indication for a biopsy to assess its capability to more accurately inform biopsy decision (Aim 3). Upon successful development, this MRF-based method will provide more quantitative tissue assessment of the prostate, optimizing biopsy avoidance in patients with a negative MRI.

NSF Awards · FY 2024 · 2024-07

The broader impact of this I-Corps project is based on the development of a technology capable of accurately and non-invasively providing diagnostic information for the identification and quantification of clinical signs and symptoms of infection and inflammation in chronic wounds. The solution will provide greater adequacy of wound bed preparation. This technology consists of sensors for real-time, non-invasive, point-of-care diagnostics for tissue evaluation in clinical settings, with a particular focus on the healing of chronic wounds. Ultimately, this technology has the potential to allow clinicians to directly correlate particular medical interventions with specific cellular responses, which could significantly improve patient outcomes and quality of life. This I-Corps project utilizes experiential learning coupled with a first-hand investigation of the industry ecosystem to assess the translation potential of the technology. The solution is based on the development of a semi-synthetic scaffold functionalized with molecular biosensors capable of providing ultra-sensitive, non-invasive monitoring of cell function. This monitoring allows physicians to tackle one of the recent trends in medical diagnostics, which is the development of point-of-care methods to enable rapid, self-supported testing in outpatient or remote settings to complement standard clinical diagnostics. The biosensors are based on aptamers, which have several advantages over antibodies and provide a higher throughput than the currently used common intermediate or end-point destructive assessments (e.g., histology and mass spectroscopy). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Osteosarcoma (OS) metastasis is the leading driver of mortality, but there are no therapies tailored to metastatic disease. Despite the development of numerous treatment modalities targeting the primary tumor, 40% of pa- tients still die from metastatic progression. Developing targeted therapies for metastatic OS has been compli- cated by extensive genomic rearrangements that differ across patients, but a common feature is that OS tends to metastasize to the lung. There is a pressing clinical need to determine the factors responsible for lung me- tastasis in OS to facilitate development of novel antimetastatic therapies. Previous findings have demonstrated the importance of alterations in enhancer activity and specific transcription factors in activating genes necessary for metastasis. However, recent studies on epigenetic subtypes of OS suggest that many of the models studied previously diverge from the epigenetic subtype of this disease most common and most deadly in the clinic. Here we propose in aim 1 to study the transcription factors (RUNX2 and SP7) that are specific to the subtype of OS that is most clinically relevant, and we plan to characterize how these create: de novo enhancer activation, novel loops and clusters in cis and trans, and novel enhancer-gene connections that promote lung metastasis. Our goal is to define “metastasis enabling circuitry”. Aim 2 tests the hypothesis that the process of lung coloni- zation is driven by an interplay between RUNX2 and signaling from the lung microenvironment through a longi- tudinal series of dynamic chromatin state changes, each with unique gene dependencies. We seek to understand the biology of the metastatic process by charting the chromatin state transitions and dependencies in osteosar- coma cells growing within the in vivo context of the lung microenvironment. If successful, we hope to expand the current arsenal of OS treatments beyond coverage of the primary lesion, to target tumor metastases during lung colonization.

NIH Research Projects · FY 2025 · 2024-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Our School of Medicine launched the first MD-PhD training program in 1956, and the program has a long history of innovation and success with many prominent alumni, including two Nobel laureates. Our mission is to develop MD-PhD training to train a multi-disciplinary pool of physician-scientist leaders to meet future biomedical research workforce needs for research leaders to accelerate research to advance the understanding, detection, treatment and prevention of human disease. Objectives include development of physician-scientists with deep expertise in one or more biomedical scientific disciplines coupled with a broad understanding across biomedical disciplines; skills to independently design and develop research programs; ability to use clinical insights to inform research; and commitment to scientific integrity. Program goals include MD- PhD completion with an efficient time to degree (TTD), publication and grants attainment productivity, post-graduation placement in training programs related to physician-scientist career development, and long-term attainment of productive research careers. Our aims include development of an MD-PhD curriculum that integrates research and clinical training with deep research experiences from the beginning of the program to promote retention of trainees in the research mission and foster curricular efficiency. We will develop required and elective MSTP activities that promote the program objectives, including didactic, research, mentoring and career development elements (RCR, RRR, grantsmanship, research communication and others) and program activities (MSTP Retreat, MSTP dinner seminars, MSTP visiting physician-scientist career sessions). We will work with our research training community to improve PhD programs and training of research mentors in best practices. Our approach includes integration of research and clinical training within each of the three basic phases: first two years (M1-M2), PhD phase (e.g. G1-4), and last two years (M3-M4). The curriculum has flexibility to meet the differing interests and needs of individual students. A key strategy is early and deep engagement with research to promote research career development and retention - in M1-M2, students complete a research rotation or graduate course in every semester in combination with the MD curriculum. Almost all students match into their PhD lab by fall of M2, allowing early launch of PhD research in M2. This is a key distinction for the CWRU MSTP and provides unique curricular opportunities. We will establish a culture of support for students and engagement of student leadership in MSTP Council to shape the program to support student professional development and wellness. We will continue to admit 12-15 students per year. The integrated research and clinical curriculum, program activities, and support for students will produce a highly skilled physician-scientist workforce with a wide range of experiences and scientific/clinical expertise, which will contribute to national goals for basic, translational and clinical research in academia, government and industry.