Ohio State University

NIH Research Projects · FY 2025 · 2017-09

PROJECT SUMMARY In eukaryotic cells, gene expression is regulated at multiple levels, including post-transcriptional gene silencing, where microRNAs (miRNAs) bind to complementary target RNAs and cause translational repression. Argonaute (AGO) proteins and miRNAs form RNA-induced silencing complexes (RISCs), the core players in gene silencing. Humans have four AGO proteins, AGO1-4, which share a high sequence identity, and the majority of miRNAs bound are common across all AGOs. Therefore, it has been thought that the four AGOs work redundantly. Nevertheless, an increasing number of studies have found that each AGO has its unique roles in various biological processes and diseases in addition to gene silencing. Although the interaction of all four AGOs with miRNAs has been well characterized, little is known about how each RISC recognizes its target RNAs. Elucidation of this recognition will provide insight into the unique roles of each AGO. Meanwhile, characterization of RISC and target interactions will facilitate target prediction accuracy by improving prediction algorithms, which will take account of not only the complementarity between guide and target but also the type of AGO and target interaction. In this proposed study, we will pursue the following specific aims. In Aim 1, we will use cryo-electron microscopy and X-ray crystallography to determine the structures of all four homogenously purified RISCs with the same guide and target RNAs, which will provide insight into the differences in target recognition by the four AGOs. In Aim 2, to clarify these differences, we recently developed a novel SHAPE-based technique which allows us to visualize the conformational dynamics of target RNA bound to RISC. The method will enable us to characterize this interaction within the RISC binding channel and its periphery at a single-nucleotide resolution and can be expanded to understand how RISCs recognize guide- binding sites buried within highly structured target RNAs. In Aim 3, we will first use mass spectrometry to identify the unique protein binding partners of each AGO and their specific sites of interaction. Then, we will use tandem immunoprecipitation, followed by RNA sequencing, to determine how the binding of these proteins influences the target specificity of each AGO and directs their functionality towards alternative cellular events. The outcome from this study will provide a solid foundation for fields beyond gene silencing and enable the development of new strategies for higher accuracy guide-RNA drug design in therapeutic applications.

NIH Research Projects · FY 2026 · 2017-07

PROJECT SUMMARY Atrial fibrillation (AF) is a leading cause of stroke and an increasingly prevalent arrhythmia in the United States due to an aging population with predisposing comorbidities (e.g. heart failure, obesity, diabetes, high blood pressure, etc.). Although there have been great technological advances in the treatment of AF, current therapies still remain insufficient due to limited understanding of the mechanisms that drive and maintain AF. Clinical studies lack reliable functional and structural mapping approaches necessary to resolve the patient-specific arrhythmogenic transmural substrate, due to the highly complex 3D structure of the human atria. Subsequently, there remains a significant debate around the mechanisms driving AF, the cause of these drivers, and how best to locate and treat these patient-specific drivers that can occur in both left and right atrial chambers. Therefore, our proposal aims to develop a novel, paradigm-shifting translational framework that uses ex-vivo to in-vivo 3D multimodal imaging approaches to accurately define disease- and sex-specific bi-atrial arrhythmogenic substrates of AF drivers, so that we can elucidate the “fingerprint” features of AF drivers. Our preliminary data led us to hypothesize that disease- and sex-specific fibrotic cellular and extracellular remodeling is heterogeneously present in one or more LA and RA transmural layers (sub-epi, intramural, and sub-endo) and can form arrhythmogenic substrates for localized reentrant AF drivers and represent personalized targets for AF treatment. We will test this hypothesis, directly in explanted human atria and a preclinical animal AF model, by integrating transmural optical mapping, clinical multi-electrode mapping, 3D MRI and PET/CT imaging, and proteo-transcriptomic analyses to define chamber- and disease-specific signaling pathways and structural- molecular-genetic fingerprints of arrhythmogenic AF driver substrates. The validated AF driver substrate fingerprints will be used to train machine learning algorithms to define patient-specific targets in persistent AF patients for either substrate modulating ablation of reentrant tracks (SMART) or anti-fibrotic therapeutic interventions. This translational research is a critical step toward the development of new personalized, mechanism-based, and sex-specific AF treatments whereby driver substrates can be accurately defined, targeted, and successfully treated to cure the most common arrhythmia in the United States.

NIH Research Projects · FY 2025 · 2017-07

PROJECT SUMMARY Cardiovascular disease remains the number one cause of death in the United States. The efficient development of future novel diagnostics and therapies to identify and treat cardiovascular disease will require highly collaborative and multi-disciplinary teams consisting of the best basic, translational and clinical investigators. To achieve this overarching goal, it will first be essential for cardiovascular researchers to possess multi-disciplinary skills and work in highly inter-disciplinary, collaborative and translational teams to efficiently move knowledge from the bench to the bedside, to the community and back. Second, to obtain optimal productivity towards improving cardiovascular health of the population, it is essential to utilize the contributions from the best of the entire scientific workforce. We have established a Training Program addressing these two unmet needs that will ultimately be required to optimally train and retain the next generation of the best cardiovascular investigators. The goals of this Training Program are: To provide multi-faceted cardiovascular research training to work in collaborative, multidisciplinary teams that will facilitate efficient translation of scientific findings to improve human health. To enhance the numbers of outstanding researchers and leaders by providing professional development skills training to promote the success and retention of the best investigators in independent scientific careers in cardiovascular research. In addition to advanced Cardiac Physiology and Biostatistics coursework, Rigor and Reproducibility, and Responsible Conduct of Research Training, several innovative Program Activities were implemented in the first funding cycle to achieve these goals. Highly interactive Professional Development Workshops supply predoctoral trainees with key professional and leadership skills to accelerate their career trajectories and retain the best scientific investigators at later career stages. These workshops also provide valuable networking opportunities for trainees with prominent visiting cardiovascular scientists. The Clinical Mentorship program allows trainees to learn the language and culture within clinical medicine and to develop the skills needed to form future collaborations with physicians. Molecular and Cellular Cardiophysiology research-in-progress presentations are highly interactive and improve both the research and presentation skills of the trainees as well as emphasize rigor and reproducibility. In this renewal, we will continue to expand upon these programs by also including Career Mentorship to meet the individualized career goals of trainees. At The Ohio State University, we have a highly collaborative and multi-disciplinary scientific infrastructure that has been created to support translational cardiovascular research, education, and training. The mentors on this T32 application are all established scientists in their field, have a passion for training, and are committed to the goals of this Training program.

NIH Research Projects · FY 2026 · 2017-07

Abstract The small Ca binding protein, calmodulin (CaM), either directly controls, or at minimum, modulates every functionality within the heart. This versatile and ubiquitous Ca binding protein is the only known exception to the central rule of biology - one gene one protein. In all mammalian cells, an identical CaM protein is derived from three different genes, CaM1,2 and 3. Finding the reason for this deviation is not only important to explain this biological conundrum, but for understanding the vast signaling processes mediated by CaM in various cell types including cardiac myocytes in health and disease. CaM is known to bind and regulate hundreds of target proteins. Due to the fact that the free concentration of CaM is limited within cardiac myocytes, yet the bound protein is abundant, we surmise the presence of the three different genes is important for orchestrating specific and unique myocyte CaM-mediated Ca signaling processes in space and time. We propose the divergent 5’ and 3’ UTRs of the three CaM genes recruit different RNA binding proteins used to transport and pool together specific CaM target proteins’ mRNAs into discrete “interactosomes”. We hypothesize these mRNA clusters are then locally translated together (“tranlatosomes”) where the proteins function within the cardiac myocyte and are thus “functionally distributed”. We have created novel imaging tools and data analysis software in order to visualize these processes. We have also discovered in the adult cardiac myocyte that ribosomal translocons, rough ER, and the golgi complex actively process and translate transmembrane proteins far from the perinuclear space, contradictory to the long held classical view of membrane protein translation. In this proposal, we will define the abundance, subcellular distribution, sites of translation and physiological modulation for the three CaM mRNAs, along with key CaM targets in cardiac myocytes in health and disease. Ultimately, we will apply this novel and new knowledge to target our therapeutically engineered proteins with high precision and specificity to select “interactosomes” in order to treat various cardiovascular diseases.

NIH Research Projects · FY 2025 · 2017-07

Abstract: Ischemic stroke affects a substantial number of people leading to long-term disability or death. Despite the prevalence of stroke, few treatment options are available. During an ischemic stroke, blood flow to a region of the brain is restricted preventing delivery of oxygen and essential nutrients. This leads to a complex environment within and around the infarcted tissue. This includes rapid cell death, inflammation, and endoplasmic reticulum (ER) stress in the surviving cells. The impact of ER stress on astrocytes and microglia and the resulting functional outcomes are unknown. ER stress activates the serine/threonine kinase protein kinase R-like ER kinase (PERK) which phosphorylates the eukaryotic initiation factor 2α (eIF2α) to attenuate protein translation. Our previous work has shown that PERK also activates the tyrosine kinase Janus kinase 1 (JAK1) to drive inflammatory gene expression. Additionally, our preliminary data show that PERK-dependent attenuation of protein translation enhances cytokine-induced inflammation in astrocytes and enhances neuronal death. Based on these findings, we hypothesize that PERK and JAK1 signaling contribute to worse stroke outcome and that targeting translational repression may provide therapeutic benefit. In this cycle, we will test this directly by deleting PERK in astrocytes and microglia to examine how PERK contributes to outcome in the stroke model of middle cerebral artery occlusion (MCAO). We will examine how translational suppression and JAK1 contribute to neuronal death and inflammation. Additionally, we will use the small molecule ISRIB to restore protein translation following MCAO to examine effects on functional outcome. We anticipate that deletion of PERK or restoration of protein translation will improve outcome following MCAO. Thus, identifying this signaling axis as a potential therapeutic target and furthering our understanding of glial biology.

NIH Research Projects · FY 2025 · 2017-07

Project Summary MicroRNAs (miRs) are evolutionally conserved small non-coding RNA molecules that are broadly involved in regulating most biological events; previous studies have focused on the canonical mRNA interference (RNAi) mechanism of miRs. During the previous funding period, we were the first to unveil an evolutionarily- conserved novel biophysical action for miRs beyond its RNAi mechanism. Specifically, we revealed a novel biophysical action of miR1, which is the most predominant miR in the heart and is downregulated in human heart failure. We found that miR1 physically binds to an inward rectifier potassium channel Kir2.1, directly suppresses the IK1 current and biophysically modulates cardiac cellular electrophysiology. Importantly, we found that a human single nucleotide polymorphism (hSNP) of miR1–– hSNP14A/G (rs776480338), in which the 14th nucleotide “A” is mutated to “G”, is a RNAi-only variant that specifically abolishes the biophysical action while maintaining the RNAi function of miR1, validating that the biophysical modulation is independent of RNAi. Our discoveries suggest that miRs modulate cardiac homeostasis through two different mechanisms: 1) canonical RNAi that regulates the expression of proteins, including ion channels, and 2) newly-discovered mechanism of direct binding with proteins that quickly results in functional modulation. With this important new finding, it is now imperative to investigate if multiple cardiac ion channels are biophysically modulated by miRs and to elucidate the specific physiological impact of miR1’s biophysical action in the regulation of cardiac (electro)physiology. Based on our published findings and preliminary data, we hypothesize that the biophysical modulation of cardiac ion channels by miRs is a general regulatory mechanism that exists broadly and plays a critical role in the homeostasis of the heart. We will study this with the following specific aims. 1) To investigate the biophysical modulation of cardiac ion channels by miR1, 2) To understand the physiological impact of miRs’ biophysical action on the heart, 3) To unveil the general mechanisms guiding miRs’ biophysical modulation of cardiac ion channels. In addition to a broad range of cellular activities regulated by the large number of miRs (>30,000 miRs in >200 species) and ion channels, our study will significantly and innovatively expand the biological significance of miR biology and ion channel biology with broad implications. Our discoveries have pioneered a new field in miR biology and will provide a mechanistic foundation and new avenue of RNA-medicine development for antiarrhythmic therapy.

NIH Research Projects · FY 2025 · 2017-06

PROJECT SUMMARY Tau aggregation is a shared pathology of Alzheimer's disease and related dementias known as tauopathies. Despite commonalities in aggregation mechanism, each disorder develops unique filamentous morphologies that reflect differential vulnerability of their constituent cell populations. In addition to providing clues about the cellular conditions associated with tauopathic neurodegeneration, the appearance of unique polymorphs in each tauopathy implicates prion-like mechanisms of templating and spread that may influence the course and severity of disease. As a result, aggregate polymorphism is a potential target for therapeutic intervention as well as for differential diagnosis of tauopathies through development of whole-brain imaging methods. This proposal seeks to advance these goals by clarifying mechanisms underlying aggregate nucleation, propagation and interaction with small-molecule probes using biochemical and structural biological approaches. Specifically it aims to (1) determine the effects of aggregation inducers including tau phosphorylation on polymorph formation, (2) clarify mechanisms through which nucleation sequence motifs common to all tau isoforms trigger aggregation, (3) rigorously test whether tau polymorphism can propagate through prion-like seeding mechanisms, and (4) identify molecular interactions that mediate the binding affinity and selectivity of tau-aggregate directed ligands. Accomplishing these goals will clarify the molecular basis of tauopathy pathogenesis and catalyze efforts toward creating novel diagnostic strategies tailored toward individual tau aggregate polymorphs.

NIH Research Projects · FY 2024 · 2017-05

Project Summary The equipment supplement funds will facilitate parent grant GM122448 (A.K. Hopper, PI) by supporting purchase of an Integra Mini 96-channel pipetter which will greatly facilitate original proposed work in Aims 2 and 3. GM122448 focuses on tRNA biology and its subcellular trafficking. tRNAs are small noncoding RNAs that are essential for decoding the genome by delivering amino acids to translating ribosomes according to codon directions in mRNAs. Defects in tRNA biology cause numerous human disorders from metabolic diseases, to neuromuscular diseases, and to cancer. tRNA biology requires a complex set of conserved gene products for post-transcriptional processing, subcellular traffic, and intron turnover. The research program impacts upon multiple facets of gene expression, quality control, and issues important to human health. We employ budding yeast and in vivo technologies to discover unknown important aspects of tRNA biology. In Aim2 of GM122884 we study trafficking of tRNAs between the nucleus and the cytoplasm. Although for decades it was thought that tRNA movement is unidirectional, nucleus to cytoplasm, we co-discovered that tRNAs move bi-directionally between the nucleus and the cytoplasm (Shaheen & Hopper 2005) and that the dynamics are conserved between yeast and vertebrate cells (Shaheen et al. 2007). We developed a new methodology, the HCl/aniline assay (Nostramo et al. 2020), that reports tRNA retrograde nuclear import and re-export to the cytoplasm. We are employing this methodology in a genome-wide screen of ~6000 yeast genes to identify and characterize the proteins functioning in the tRNA retrograde pathway. Aim 3 of GM122884 investigates tRNA introns. Possession of tRNA introns in subsets of tRNA genes has been conserved from Archaea to humans. We recently made the exciting discovery (Nostramo et al., Mol. Cell, in revision) that introns spliced from pre-tRNAs provide a novel complementary-dependent mechanism to fine-tune basal mRNA levels and to assist cellular responses to stress. We also learned that under particular stresses, subsets of tRNA introns accumulate to high levels due to increased stability. We discovered one mechanism for tRNA intron turnover (Wu & Hopper 2014); however, there are at least four additional unknown mechanisms to destroy tRNA introns. None of the yeast annotated RNases appear to function in the unknown turnover pathways. Thus, we are conducting a genome-wide screen for mutants that accumulate each of the 8 tRNA intron families whose turnover remains unknown. Although we are able to grow yeast mutants in the deep 96-well plates, we are unable to extract RNAs in this format due to the opaqueness of the microtiter plates; therefore, we must conduct the biochemical steps in labor-intensive analyses of single mutants. The Integra Mini 96-channel pipettes will allow us to conduct all steps preceding northern gel analyses for 96 strains simultaneously and thereby greatly reduce the time and safety to complete Aims 2 and 3.

NIH Research Projects · FY 2026 · 2017-05

Project Summary Current FDA-approved drugs are usually either small molecules (MW <500) or large proteins (MW >5000). Small molecules are generally limited to targeting proteins (and other biomolecules) that contain deep binding pockets (e.g., enzymes and GPCRs), which represent ~10% of all disease relevant human proteins. On the other hand, biologics (e.g., monoclonal antibodies) are restricted to extracellular targets, which represent another ~10% of all drug targets. The remaining ~80% drug targets, which are primarily proteins involved in intracellular protein-protein interactions (PPIs), are currently undruggable by either approach. The same limitations apply to the use of small molecules and proteins as research tools. In addition, there are ~7000 human genetic diseases affecting ~10% of the US population; only a very small fraction of them currently has pharmacologic treatment. The overall goal of my research is to develop a general approach to targeting the ~80% undruggable proteins and treating human genetic diseases. Accomplishing this goal requires effective delivery of large biomolecules into the mammalian cell. Over the past decade, my group has discovered a novel class of cyclic cell-penetrating peptides (CPPs), which efficiently deliver all major drug modalities into the cytosol of mammalian cells in vitro and in vivo, and elucidated their mechanism of endocytic uptake and endosomal escape. During the next five years, we will continue three areas of investigation. First, we will investigate how linear and cyclic CPPs directly translocate across the plasma membrane, how bacterial toxins and some human proteins escape the endosome into the cytosol, and how CPPs and some proteins exit the mammalian cell by a yet poorly defined “unconventional protein secretion” mechanism. Second, we will use the mechanistic knowledge gained to develop CPPs of improved properties, e.g., CPPs with specificity for tumor tissues, and engineer a mammalian membrane translocation domain (MTD) for intracellular delivery of proteins. Finally, we will leverage the cyclic CPPs and MTDs to develop cell-permeable peptides and proteins as chemical probes and potential therapeutics against several key “undruggable” targets.

NIH Research Projects · FY 2025 · 2017-03

ABSTRACT The purpose of the proposed renewal is to add a longitudinal component for the purpose of generating risk models to predict progression in ocular diseases that include biomechanical metrics established at baseline. A novel metric of Corneal Contribution to Stress was developed at baseline that represents long-term adaptation to asymmetric biomechanical properties in keratoconus which we propose will predict progression over the follow-up period. An additional metric of cornea compressibility will be included in the progression model. In Diabetes without retinopathy, we propose that scleral stiffness will predict development of diabetic retinopathy during the follow-up period, and that increased scleral stiffness is a cumulative indicator of long-term hyperglycemia which we attribute to the formation of non- enzymatic cross-links in the sclera. Hemoglobin A1c will be included in the model as a discrete indicator of hyperglycemia. Finally, we propose to add elastic and viscoelastic biomechanical metrics to the risk model from the Ocular Hypertension Treatment Study to predict the conversion from ocular hypertension to glaucoma. Elastic parameters include corneal stiffness and scleral stiffness, and the viscoelastic parameter is corneal hysteresis. Once completed, these risk models can be translated to the clinic as new tools to aid in the management of multiple ocular conditions. .

NIH Research Projects · FY 2026 · 2017-02

SUMMARY Cardiac dysfunction is among the most common extrapulmonary complications of severe influenza virus infections. Although the heart complications of influenza virus infection are a clearly recognized clinical problem, they are poorly studied in terms of pathogenic mechanisms. We lack basic scientific understanding of 1) whether influenza virus directly or indirectly causes heart damage, 2) what viral features facilitate cardiac tropism of certain strains, 3) how virus disseminates from the lung specifically to the heart in the absence of viremia, and 4) how the immune system influences heart pathogenesis. The major barrier to mechanistic research in this area has been the lack of tractable animal models that recapitulate significant cardiac dysfunction in severe influenza. We have overcome this obstacle by developing interferon-induced transmembrane protein 3 (IFITM3) KO mice as a model of severe influenza virus infection that includes viral replication in the heart and significant cardiac electrical dysfunction, inflammation, and fibrosis. Importantly, IFITM3 is an antiviral protein of the innate immune system in which common deficiencies in the human population render individuals more susceptible to severe infections, making IFITM3 KO mice a relevant and informative model. We will make use of this groundbreaking model in the following three Aims. In Aim 1, we will determine whether virus dissemination and replication directly in heart tissue is required for this pathology or whether influenza virus indirectly induces heart dysfunction through systemic inflammation from the severely infected lung. To address this fundamental controversy in the field, we have developed a novel and innovative recombinant influenza virus strain with specific inability to replicate in heart cells while being fully replicative in the lung. In Aim 2, we will identify viral features that endow specific virus strains with cardiac tropism by using a candidate viral gene approach with reassortant viruses, as well as an adaptive passaging approach. In Aim 3, we will solve the mystery of how influenza virus moves specifically from the lung to the heart in the absence of viremia and other organ infections. For this, we will test whether infection of migratory immune cells facilitates virus trafficking to the heart. We will further investigate how IFITM3 expression in hematopoietic immune cells influences cardiac dissemination of virus and cardiac dysfunction during infection. Overall, our research will answer fundamental questions in the influenza field, and will reveal new strategies for combatting influenza-associated cardiac dysfunction.

NIH Research Projects · FY 2026 · 2017-01

Endothelium-derived nitric oxide (NO), is a key mediator regulating vascular tone and blood pressure. NO mediates vascular relaxation through activation of soluble guanylate cyclase (sGC) in the smooth muscle. While NO is synthesized by NO synthase in the endothelium, the process of vascular NO degradation and metabolism in smooth muscle is poorly understood. NO degradation in the vessel wall is mediated by an O2- dependent NO dioxygenase (NOD) that oxidizes NO to nitrate. Cytoglobin (Cgb) is a recently discovered globin expressed in smooth muscle (but not in the endothelium) with previously unknown function. Cgb is proposed to serve as a critical regulator of the rate of O2-dependent NO metabolism in the vessel wall, in turn regulating vascular tone. Over the prior grant period: 1) we demonstrated that Cgb is the major heme protein that regulates the rate of O2-dependent NO metabolism in the smooth muscle of both conduit and resistance vessels and, in turn, profoundly modulates vascular tone; 2) we identified that cytochrome b5 (B5)/ B5 reductase (B5R) constitutes the major Cgb reducing system that supports this NOD function; 3) we discovered that Cgb has potent superoxide dismutase (SOD) function accounting for its previously unexplained antioxidant effects; 4) most recently, we identified novel selective inhibitors of Cgb NOD function that do not impair its SOD function and were shown in vessels to enhance NO mediated sGC activation. However, major questions remain regarding the overall process of NO decay in the vessel wall, how this varies in disease, and how it can be modulated to ameliorate disease. In the next stage of this grant program, we seek to determine how the NO degrading and SOD/antioxidant properties of Cgb in the vessel wall control the processes of NO and redox metabolism in normal and hypertensive vessels, and how this in turn regulates vessel tone and systemic blood pressure. The critical effects of Cgb expression levels and the modulation of its NOD activity will be determined. Studies will be performed first in isolated vessels and then in the in vivo cardiovascular system, with measurements in our genetically modified mouse lines with Cgb-/-, Cgb-/+ and Cgb overexpression as well as compounds that selectively inhibit Cgb NOD function. Studies will also be performed in our recently developed conditional smooth muscle-selective Cgb-/- mouse to definitively characterize the role of Cgb in smooth muscle. Studies will focus on the physiological regulation that occurs in normal non-hypertensive mice and then on angiotensin-induced hypertension. All the data obtained will be used to support computational modeling that will enable us to predict the effects of modulating Cgb expression and its NO metabolizing function. Accomplishment of this research plan will elucidate how Cgb levels and its NOD and SOD function regulates O2-dependent NO metabolism and the redox state of the vessel, thus providing important insights into the regulation of vascular tone in normal physiology and cardiovascular disease. This knowledge will lead to new therapeutic approaches to treat or reverse hypertension and other cardiovascular disease.

NIH Research Projects · FY 2025 · 2016-12

PROJECT SUMMARY / ABSTRACT New HIV-1 infections continue to drive a worldwide pandemic. Combinatorial anti-retroviral therapies (cART) have helped to blunt the clinical outcomes of HIV-1 infected individuals. However, drug-resistance mutations continue to challenge cART regimens underscoring the importance of identifying new viral drug targets. HIV-1 integration into the human genome is essential for a productive infection. Integration is catalyzed by the retrovirus encoded integrase (IN) that forms a complex with the long terminal repeat (LTR) ends of the viral cDNA, produced by reverse transcription of the HIV-1 genomic RNA. The resulting intasome precisely positions the LTR-ends for catalytic strand-transfer into a genomic target site. Structural comparisons show that all seven retrovirus genera maintain a central Conserved Intasome Core (CIC) containing a tetramer of IN. Some retrovirus family members expand the structure surrounding the CIC by appending additional IN subunits. For example, the prototype foamy virus (PFV) intasome assembles into a simple IN-tetramer while the mouse mammary tumor virus (MMTV) forms an IN-octamer by attaching IN-dimers to either side of the CIC. IN octamer, decamer, dodecamer (12-mer) and hexadecamer (16-mer) intasomes have been reported for HIV-1. Remarkably, the contributions of IN-multimer architecture to HIV-1 biology is largely unknown. The IN-assembly progressions that result in a fully formed HIV-1 intasome are similarly unknown. During infection, reverse transcription and intasome assembly occurs at or near the nuclear membrane. The host cofactor LEDGF/p75 appears to facilitate chromatin localization of the HIV-1 intasomes, and deletion of LEDGF/p75 reduces HIV-1 integration at least 10-fold. We have found the non-conserved peptides linking well- known conserved IN domains control HIV-1 IN-multimer architecture, and that LEDGF/p75 is necessary for efficient HIV-1 intasome assembly in vitro. These observations underpin several key unanswered questions: What are the factors that guide IN multimer progressions resulting in a fully assembled HIV-1 intasome? What is the function of LEDGF/p75 in HIV-1 intasome assembly and/or chromatin interactions? How does HIV-1 IN- multimer architecture impact genomic target site selection in cellulo? We propose to utilize innovative real-time single molecule imaging and analysis to understand the contributions of IN-multimer architecture on HIV-1 mechanics with the following Specific Aims: 1.) determine the IN assembly progressions that control HIV-1 intasome architecture, 2.) determine the role of HIV-1 intasome architecture on the dynamic interactions with defined target DNA and chromatin in vitro, and 3.) determine the role of HIV-1 intasome architecture on targeting host chromatin features in cellulo. These studies are designed to interrogate the animated processes that support HIV-1 intasome architecture with the goal of identifying additional retroviral progressions that might be exploited as therapeutic targets.

NIH Research Projects · FY 2025 · 2016-09

The goal of this proposal is to further evaluate the safety and feasibility of gene transfer to provide aromatic L- amino acid decarboxylase (AADC) enzyme into the midbrain of patients with AADC deficiency and continue Biologics License Application (BLA)-enabling studies as per FDA recommendations. AADC deficiency is a devastating genetic neurometabolic disorder which causes hypotonia, dystonia, intense and long-lasting oculogyric crises (OGC), developmental delay and chronic and severe neurological dysfunction. A gene therapy based on delivering of a recombinant adeno-associated virus carrying the DDC human gene (AAV2-AADC) to the brain structures that physiologically AADC enzyme (midbrain) could be a most needed disease-modifying treatment for AADC deficiency. Eight (8) AADC deficient patients have been treated (160 µL) in our initial NIH- funded trial in the US under BB-IND-16127 and an additional 15 subjects under an ethics committee-approved compassionate use program (CUP) in Poland. The latter received a larger infusion volume (≤300 μL) and a shorter surgical procedure. Both approaches were safe and well-tolerated regardless of dose or volume of infusion. OGCs stopped a few weeks after the surgery and subjects’ sleep, mood and irritability improved. Most subjects are gaining head control and muscular tone, developing purposeful movements and some are even sitting up and starting to walk without support, regardless of their age. Encouraged by the safety and positive biomarker and clinical outcomes observed in those groups, we propose an extension of the BB-IND-16127 study to (i) determine the long-term (up to 5 years) safety and tolerability of the surgical infusion of already treated subjects (n=8) (ii) determine the safety and tolerability of a larger volume of Cohort 2 vector concentration into the SN/VTA administered via a surgical procedure optimized to increase safety by reducing surgical and anesthesia times (single-cannula insertion per hemisphere) in AADC deficient patients >4 years, and (iii) demonstrate effective restoration of AADC function by measuring CSF neurotransmitter metabolites and changes in brain FDOPA uptake on PET imaging. This will be a multi-center study with subjects to be treated at The Ohio State University and at the University of California San Francisco. As per our discussions with FDA, the study design includes a 12-month lead-in period that will serve as a natural history control group to explore potential efficacy of this novel treatment. Cohorts 3 (4-13 years, n≤12) and 4 (>13 years, n≤12) will then receive a larger infusion volume of AAV2-AADC at the same titer Cohort 2 received (2.6 x 1012 vg/mL; up to 300 μL/hemisphere). Renewal of funding for this trial will enable assessment of the safety and tolerability of an optimized dose and delivery procedure to enhance distribution of AADC expression within the midbrain, which we hypothesize may lead to further clinical improvement. Completion of this exploratory clinical trial will pave the way to registration of this disease-modifying AAV2-hAADC gene therapy for AADC deficiency and future gene therapies for other neurological disorders.

NIH Research Projects · FY 2026 · 2016-09

Project Summary Epithelial cells, which line both the inside cavities and outside of the body, exist in tissues as monolayers, multilayers of cells, and three-dimensional tubular, ductal, or glandular structures. Within these structures a principal function of epithelial cells is to form a contiguous barrier to restrict movement of ions, water, macromolecules, and pathogens. During developmental and regenerative processes, epithelial cells must undergo morphogenetic processes to create and organize tissues, including branching morphogenesis and luminogenesis. Proper formation and homeostasis of the epithelium is critical for tissue and organ function; dysregulation of the epithelium is associated with epithelial barrier loss (including sepsis), defective wound healing, and development and progression of cancer. This NIGMS R35 MIRA renewal is focused around three main research goals that will each examine how biophysical mechanisms, including mechanical forces, regulate epithelial developmental and homeostatic processes. In Research Goal 1, new force biosensors will be used to understand how keratin and vimentin intermediate filaments (IF) experience mechanical forces, and how these forces regulate cellular processes such as migration. Additional studies in Goal 1 will focus on understanding how desmosomes, IF-connected cell-cell adhesions, regulate branching morphogenesis and stabilize epithelia during pressure-driven mechanical loading of tissues. In Research Goal 2, the role of the nuclear LINC complex in regulating epithelial homeostasis will be examined through studies to uncover the mechanisms by which the nuclear LINC complex regulates RhoA activity and actomyosin contractility. In addition, studies of mammary glands with and without the LINC complex will be performed to understand how the nuclear LINC complex contributes to epithelial homeostasis. Research Goal 3 is focused on the biophysical factors that contribute to formation and stabilization of three-dimensional epithelial cell structures. A new chloride biosensor will be used to understand how apical chloride secretion changes during luminogenesis and if chloride secretion is dynamically regulated to stabilize the lumen. Next, stretch activated ion channels, including Piezo1, will be examined to determine how these mechanosensitive channels regulate luminogenesis and lumen coalescence. The final focus of this goal is to understand how apical-basal polarity inversion enables migratory phenotypes. This comprehensive study of epithelial biophysics, which including the forces across cell-cell adhesions and nuclear forces, will continue to advance the understanding of how epithelial homeostasis is regulated, which is relevant to the processes of wound repair, inflammation, and epithelial tissue development and organization, as well as epithelial diseases, including cancer, fibrosis, and chronic inflammation.

NIH Research Projects · FY 2025 · 2016-08

Project Summary/Abstract Chiral amines are prevalent motifs in nature and medicine. Conversely, alcohols are among the cheapest and most ubiquitous molecules. This program is dedicated to the development of new, remote C-H functionalization strategies that will streamline the synthesis of biologically relevant amines from abundant alcohol precursors. The key innovation of the proposed approach is the design of catalysts to harness regioselective 1,5-hydrogen atom transfer (HAT) mechanisms via imidate (β) and amidyl (δ) radicals. Through catalyst-controlled generation of nitrogen-centered radicals, as well as newly enabled regulation of their remote (β or δ) terminations, we seek to access new types of reactivity and selectivity. For example, the first asymmetric examples of several radical-mediated transformations are proposed. Additionally, a series of novel, single and double C-H functionalization cascades are described. The elucidation of these divergent mechanisms for intercepting radical intermediates will enable new access to valuable, remote transformations, including the synthesis of several medicinally relevant scaffolds (e.g. α-amino acids, β-amino alcohols, aza- heterocycles) in a rapid, modular, and selective fashion. The significance of these strategies for radical-mediated, remote C-H functionalization is their facilitation of the discovery of otherwise unlikely synthetic reactivity. Specifically, by employing transient imidate radical chaperones, alcohols may be selectively converted to a family of chiral amines. Two metal-catalyzed classes of reactivity (stereoselective HAT and termination, or desymmetrization by HAT) are proposed to facilitate asymmetric β C-H amination and other enantioselective functionalizations. In parallel, new approaches for metal-mediated, amidyl radical generation from amines will facilitate their more rapid and direct access, as well as new avenues for their remote cross-coupling with arenes and other nucleophiles. The newly elucidated mechanisms for harnessing novel δ C-H reactivity (by amidyl radicals) will also enable further developments of chaperone-mediated β C-H functionalizations (by imidate radicals). Together, these studies will further expand our ability to selectively control remote HAT mechanisms and enable novel, useful, and unprecedented C-H editing of alcohols and amines found in medicinally relevant molecules.

NIH Research Projects · FY 2025 · 2016-08

Project Summary Cytokinesis is essential for cell proliferation, cell differentiation, and tissue homeostasis. Most events and proteins of cytokinesis are conserved from yeast to humans. Majority of the proteins involved in cytokinesis have been identified, however, the mechanisms of actomyosin contractile-ring constriction, plasma-membrane expansion in the cleavage furrow, and extracellular matrix remodeling are still poorly understood; nor do we understand how cells coordinate these events for successful cell division. Here we propose to continue elucidating the molecular mechanisms of cytokinesis using fission yeast as a model system. Previous studies and our solid preliminary data led to the central hypothesis of this proposal that coordination between exocytosis and endocytosis is essential for successful plasma membrane deposition and septum formation during cytokinesis, and septins and Ync13 are among the crucial coordinators. We will use complementary genetic, cellular, microscopic (confocal, TIRFM, electron microscopy, electron tomography, and super-resolution), biochemical, structural, and computational approaches to test this hypothesis by investigating three specific aims: 1) Elucidate how septins define the sites of vesicle tethering by the exocyst and how they affect the sites of endocytosis; 2) Characterize how Ync13 coordinates exocytosis and endocytosis during cytokinesis; 3) Investigate molecular mechanisms of trafficking, anchoring, and regulation of glucan synthases during cytokinesis. Our studies on the relationships between septins, the exocyst, Munc13/UNC-13 protein Ync13, F- BAR protein Rga7, coiled-coil protein Rng10, and glucan synthases will provide molecular links among the main cytokinesis events. Our proposed studies are significant because they will advance the understanding of cytokinesis in three important ways: a) What are the roles of septins in exocytosis and endocytosis during cytokinesis; b) how Ync13 coordinates exocytosis and endocytosis for successful cytokinesis; c) how septum synthases are trafficked and regulated. The concepts learned from this project will be applicable to understand the coordination of septins, the exocyst, plasma-membrane deposition, and extracellular matrix remodeling in human cells because our three specific aims involve the most conserved aspects of cytokinesis. Because the fungal specific essential enzymes such as glucan synthases, which build the septum during cytokinesis, are targets of several antifungals, our studies on the regulators of these synthases proposed here may lead to novel targets for antifungal drugs. Thus, our discoveries on both conserved and fungal-specific aspects of cytokinesis may be harnessed to improve human health.

NIH Research Projects · FY 2025 · 2016-06

PROJECT SUMMARY Little is known about the role of micromechanical stress in regulating morphology and function of neurons in the central nervous system (CNS). Axonal varicosities (swelling or beading) are enlarged, heterogeneous structures along axonal shafts, profoundly affecting axonal conduction and synaptic transmission. They are a key pathological feature believed to represent slow accumulation of axonal damage that occurs during irreversible degeneration, for example in mild traumatic brain injury (mTBI), Alzheimer’s disease (AD), and multiple sclerosis. In the first funding period of this R01, we discovered that fluid mechanical stress immediately and reversibly induced varicosities in unmyelinated axons of cultured CNS neurons, and we further visualized varicosity induction in vivo. Most brain regions including the corpus callosum in healthy adults contain both myelinated and unmyelinated axons, while myelin appears to protect the axon from initial mechanical injury. Using a mouse model mimicking concussion, our new studies have found immediate varicosity formation in unmyelinated axons of cortical neurons and delayed demyelination in the cortex after mechanical impact. Our new results have also indicated that microtubule (MT)-associated protein 6 (MAP6) regulates axonal varicosity formation through its properties of MT stabilization and Ca2+/calmodulin binding. Based on our new findings, we hypothesize that mechanical impact immediately induces varicosity formation in unmyelinated axons, which is restrained by MAP6-mediated MT stabilization and subsequently promotes adjacent demyelination that increases axon vulnerability to second impact, leading to a vicious cycle in repeated mTBI and hence worsened behavioral impairment. To test this original hypothesis, we will use a multidisciplinary approach including mTBI and demyelination mouse models, cell-type-specific overexpression, knockout and rescue, confocal and electron microscopy, behavioral testing, electrophysiological recording, a versatile biomechanical assay, myelin coculture and state-of-the-art imaging techniques. We will determine (Aim 1) whether mTBI-induced axonal varicosity formation and behavioral impairment can be aggravated by MAP6 deletion and ameliorated by MAP6-mediated MT stabilization, (Aim 2) how MAP6 regulates axonal varicosity initiation, recovery, location, heterogeneity and long-term fate through distinct signaling pathways in partially myelinated axons, and (Aim 3) how MAP6 regulates oligodendrocyte mechanosensation, and whether preexisting demyelination and/or preexisting axonal varicosities increase the risk of injury from repeated mechanical impact. This project represents an underexplored research field with many open questions. This research is significant because it will provide novel mechanistic insights into central neuron mechanosensation and mTBI primary injury.

NIH Research Projects · FY 2024 · 2015-09

ABSTRACT The mechanistic underpinnings of diseases involving the exocrine pancreas are poorly understood. Chronic pancreatitis is often accompanied by inflammation of the pancreas, irreversible fibrosis, and destruction of the pancreatic parenchyma resulting in abdominal pain, malnutrition, exocrine pancreatic insufficiency, pancreatogenic diabetes, and, in some cases, pancreas cancer. We have developed a network of adult and pediatric investigators to form The Ohio State University Clinical Center (OSU CC). In a multidisciplinary collaboration with other clinical centers in the Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer Clinical Centers (CPDPC-CCs) (U01), the OSU CC has helped develop four (4) major observational studies during the initial funding cycle to address research gaps in our understanding of diseases of the exocrine pancreas. Specifically, the OSU CC seeks to validate proposed diagnostic biomarkers in chronic pancreatitis, pancreatogenic diabetes, and pancreas cancer.

NIH Research Projects · FY 2025 · 2015-09

With the climate change-related emergence of new pathogens and the reemergence of old, once-controlled pathogens, as well as in light of rapidly developing antibiotic resistance, society is in urgent need of a more thorough understanding of the bacterial pathogenicity and its major mediators – toxins and toxic effector proteins (effectors). The actin cytoskeleton is a common target for numerous bacterial effectors. However, the mechanisms by which many actin-targeting bacterial toxins exhibit their pathogenicity remain poorly understood. The long-term goal of the proposal is to decipher the in-depth molecular and cellular mechanisms of bacterial effectors targeting the actin cytoskeleton to i) enable alternative ways of targeting pathogens and ii) get a deeper understanding of the actin cytoskeleton per se. The current proposal is directly relevant to the NIH mission as it focuses on three families of bacterial effectors, all produced by human pathogens. Furthermore, the proposal is of interest for a general understanding of human physiology as each of the above toxins reveals novel properties of the actin cytoskeleton. Thus, Aim 1 focuses on deciphering the novel hitherto unprecedented ability of VopF and VopL effectors produced by Vibrio cholerae and Vibrio parahaemolyticus to potentiate actin processive polymerization at the pointed ends. Aim 2 addresses the properties of SipA, an effector from Salmonella enterica that exerts a novel mode of binding to actin, bestowing the pathogen an ability to adhere to and invade the polarized host cell. In Aim 3, the proposal will decipher the mechanisms of actin-dependent membrane remodeling by Legionella pneumophila effectors. Understanding these and related processes is particularly important given that all intracellular pathogens depend heavily on hijacking host membrane organization to survive and thrive inside the affected cell. The research strategy for the toxin characterization will be based on using several highly complementary experimental approaches. The effects of the toxins on actin dynamics in bulk and at the single-filament level will be combined with cell biology and structural biology approaches. Specifically, bulk actin dynamics will be monitored via modifications of the pyrene-actin polymerization approach. The effects of the toxins on the actin dynamics at the single-filament level will be characterized by total internal reflection fluorescence microscopy, which will be enforced by microfluidics (in collaboration with Dr. Shekhar) for deciphering the mechanisms employed by VopF/VopL toxins (Aim 1). A modification of this technique will be used to decipher the mechanisms of a membrane-reorganizing protein MavH (Aim 3). The structures of VopF with the actin pointed end (Aim1), and SipA bound along the filament side (Aim 2) will be characterized by the cryoEM reconstruction in collaboration with Dr. Egelman’s group. Fluorescence anisotropy will be used to describe the strengths of the interactions between actin and the effectors. The proposed study is significant and innovative as it fills major gaps in our understanding of the toxicity of several life-threatening pathogens and reveals novel ways of operation of the actin cytoskeleton.

NIH Research Projects · FY 2025 · 2015-08

Project Summary The overarching goal of this research is to better understand the human visual system, and how objects and their locations are perceived and represented in the brain. The proposal investigates a fundamental challenge for our visual systems: Visual information is coded relative to the eyes, but the eyes are constantly moving. How, then, do we achieve spatial stability? The world does not appear to “jump” with each eye movement, but this seamless percept belies a complicated computational process. Moreover, spatial localization is not an isolated process; it interacts with attention, object recognition, depth perception, memory, cognitive control, and more. In order to understand visual stability, we need to account for both how spatial information is represented (or “remapped”) across eye movements, and how spatial information is integrated with these other processes. Research under the prior award made strong strides in two key directions along these lines: understanding how 2D spatial information is integrated with depth information to consider spatial stability in 3D, and revealing how spatial remapping impacts feature/object perception. In the next stage of this research program, we build on this momentum to investigate spatial and object stability across eye movements – and their integration more broadly – with a special focus on the roles of dynamic context and top-down attentional control. In Aim 1 we employ an fMRI-EEG fusion approach to investigate 3D stability and object integration in visual cortex and the hippocampus, hypothesizing that dynamic, active saccade context may promote enhanced visual integration and stability. Next, we further develop the PI’s Dual-Spotlight Theory of remapping (Golomb, 2019) and explore the role of top-down attentional control in remapping and perceptual stability (Aim 2). Finally, we develop a new model-based neuroimaging analysis technique to enable future progress on persistently less tractable aspects of this question (Aim 3). The proposed experiments strive to continue to transform our understanding of visual stability, particularly how it interfaces with other perceptual and cognitive processes that are central to our understanding of human perception and brain function. The research proposed here will have an immediate impact on our understanding of typical visual functioning in healthy human populations. These advances could also have a longer-term impact on a variety of clinical applications, informing our knowledge and assessment of visual disorders resulting from eye disease, injury, brain damage, and development/aging.

NIH Research Projects · FY 2024 · 2015-04

Enhanced Raman Imaging of Ligand-Receptor Recognition Abstract: The goal of this proposal is to identify molecular interactions relevant to drug targeting and chemical signaling in living cells. A critical bottleneck in the development of drugs is the identification of off-target effects. Methods that can identify the molecular interactions associated with proteins recognizing and binding to drug candidates in cellular and other live models can be used to understand and minimize unwanted side effects and complications. Identifying these effects at earlier stages of drug screening is important to avoid late stage drug failure. We are developing technologies that take advantage of the plasmonic properties of metallic nanoparticles to enable chemical-specific spectroscopic studies of ligand-receptor binding in living cells. These investigations will provide new approaches to probing the receptor’s chemical residues that bind peptide antagonists and provide insights into molecular interactions that regulate the proteins involved in signaling and drug targeting. Our approach combines enhanced Raman scattering from both nanoparticles (Surface enhanced Raman scattering, or SERS) and scanning probes (tip enhanced Raman scattering, or TERS), nanoparticle tracking microscopy, non-standard applications of static and dynamic quantum chemical calculations, and super- resolution SERS imaging to characterize chemical interactions that regulate binding to protein receptors. Information present in the enhanced Raman scattered response provides molecular level detail of the interactions governing recognition by the protein. In concert, our methodologies provide a new approach to monitoring protein binding to putative drugs in living cells, and to characterize targeting specificity. The specific aims of this proposal are: 1) Screen the targeting specificity of peptide-functionalized nanoparticles in live cells. 2) Develop super-resolution SERS imaging to improve binding specificity studies and identify particle location in cells. 3) Combine non-standard quantum and numerical simulations with experiments to identify key motifs in amino acid conformation related to peptide binding. Overall, the technology and platform we propose will address the challenge of obtaining chemical information from ligands binding to receptor proteins in intact cells. These studies will provide new insights into the molecular interactions that regulate signaling pathways and how anomalies in these interactions are associated with disease and treatment.

NIH Research Projects · FY 2025 · 2015-03

Summary Mammalian genomes are subject to a constant barrage of damage from metabolites, external agents, or physiologic processes, including transcription and replication. Developing lymphocytes also target double- strand breaks (DSBs) to antigen receptor loci during V(D)J recombination. To maintain genomic stability, DSBs must be repaired with high fidelity, minimizing oncogenic alterations such as chromosomal deletions and translocations. The DSB response extensively revises flanking chromatin via ATM-mediated phosphorylation of the histone variant H2Ax, producing γH2Ax, which spreads for 100s of kb around a DSB. In somatic cells, most of which are non-cycling, γH2Ax domains serve as chromatin-based platforms to facilitate repair by the non- homologous end joining (NHEJ) and, likely, as adherent surfaces to hold broken chromosome ends together. Indeed, ends are destabilized in cells lacking ATM or H2Ax, which have elevated levels of translocations. Thus, a deeper understanding of mechanisms that coordinate DSB repair and sequester ends from the rest of the genome remains an important goal. In this regard, links between repair, transcription, and epigenetic landscapes around DSBs are emerging. A feature that bridges many of these processes is the 3D conformation of chromatin, which determines the range of chromosomal contacts made by a persistent DSB. The applicant has shown that the topological “environment” of a DSB in non-cycling lymphocytes determines the spread and contours of γH2Ax domains, paralleling chromosome contacts of the break site. In addition, transcription of genes within γH2Ax domains was repressed, perhaps minimizing introduction of new breaks associated with RNA polymerase readthrough. A key finding from the prior funding period was that DSBs near the border of topologically-associated domains (TADs) produce highly asymmetric γH2Ax platforms on each chromosome end – one of which is very short – which may enhance disassociation of chromosome ends when the break persists. Indeed, genomic alterations, including those associated with cancer, are enriched near topological borders. Launching from these discoveries, the applicant now proposes to define the functional relationships between chromosome topology and DSB repair outcomes. Overarching hypotheses for three aims of the project are: (i) persistent DSBs adjacent to TAD borders will generate distinct profiles of repair products due to unstable association of chromosome ends, promoting extensive deletions and translocations, (ii) the mechanism of TAD formation, called loop extrusion, is required for generation of DDR platforms; impairment of this process will deleteriously affect repair outcomes, and (iii) transcription within a γH2Ax domain harboring a persistent DSB will enhance the probability of its deletional repair to an expressed gene with which it contacts. Together, the proposed project will fill fundamental knowledge gaps about how DSB responses integrate spatial, transcriptional, and chromatin-based mechanisms to sequester chromosome ends for efficient repair, minimizing their oncogenic potential in somatic cells.

NIH Research Projects · FY 2025 · 2014-09

Project Summary/Abstract: It is known that individuals with Down syndrome (DS) have reduced visual acuity which may result from high levels of refractive error (sphere and cylinder) and elevated levels of higher-order aberrations that are present from childhood. Targeting optical aberrations through spectacle correction with refractions determined objectively from measures of wavefront aberrations is part of our long-term goal to provide new treatment strategies to improve vision in this group. The central hypothesis is that measures of visual and retinal image quality (metrics) can be used to identify sphere and cylinder corrections that optimize vision, resulting in multi-line acuity gains over traditional corrections. Our previous evaluation of these corrections in adults with DS found improvement in acuity, but not the multi-line levels predicted. We propose that this mismatch in acuity gains is attributed to decades of exposure to poorly corrected optical deficits that has resulted in a reduction in visual acuity potential (i.e. refractive amblyopia). Further, the method is inherently monocular, and does not prescribe refractions reflective of the two-eyed visual experience of the patient, as would occur in a clinical exam. The short-term goal of this work is to address these proposed barriers to multi- line acuity improvements with metric-optimized refractions through a randomized treatment trial conducted in children with DS who are within the optimal age for treatment of amblyopia. This trial will compare acuity outcomes for three refractive methods: 1) metric-optimized refractions, 2) clinical refractions, and 3) a hybrid method that allows for clinical refinement of the metric-optimized refraction. In addition, we seek to improve the objective refraction process by removing dilation prior to the wavefront imaging necessary to compute the metric values, as well as determining the rate of change in refraction during extended follow-up to guide patient recall times (i.e. follow-up intervals) for updated refraction. This work will be accomplished through three specific aims: Aim 1) Compare refractions and resultant visual acuity obtained from wavefront measures pre- and post-dilation. Aim 2) Compare visual acuity outcomes of metric-optimized, clinical, and clinically-refined metric-optimized refractions in a treatment trial of children with DS. Aim 3) Determine rate of change in refraction as a function of age and refractive error type annually over four years in children and adults with DS. Metric-optimized refraction is innovative in that it compensates for the absence of subjective input in the refraction process for the DS population and allows the clinician to consider corrections targeting overall image quality. This proposed research is significant in that it proposes to use spectacles in an optimized manner to remove visual barriers. Treatment of refractive amblyopia with multi-line acuity gains would lead to lifelong benefits, including access to traditional print size, facilitate educational efforts, and facilitate activities of daily living, all of which may promote greater independence. This work is similarly applicable to other patient populations who cannot fully participate in subjective refractions (young children, cognitively impaired, etc.).

NIH Research Projects · FY 2025 · 2014-08

Project Summary This renewal Advanced Research Training in Immunology for Surgery Trainees (ARTIST) is a specialized training program that responds to the accelerating demand for expertise at the intersection of surgery and translational immunology research and the critical need to ensure a pipeline of surgeon-scientists. The long- term goal of the program is to inspire and train the next generation of surgeon-scientists who will lead immunology research focused on solutions for clinical problems relevant to tissue injury, repair, regeneration and replacement. Discoveries by these surgeon-scientists are anticipated to lead to new tools for diagnosis or prognosis of immune-mediated diseases and also to new immunotherapies. Program objectives are achieved by recruitment of outstanding trainees with innate curiosity and the aspiration to become leaders in surgery, selection of high quality interdisciplinary and collaborative NIH-funded faculty, and engagement in innovative didactics and professional development activities. Eligible trainees are competitively selected from an outstanding pool of surgery residents who have completed at least two years of clinical training in the Ohio State University (OSU) General Surgery residency program, in other surgical subspecialty residencies at OSU Wexner Medical Center and Nationwide Children’s Hospital or in general surgery residency programs at other institutions. The diversity of the surgical trainee pool is a strength of the ARTIST program and will contribute to the quality and diversity of the future biomedical surgeon-scientist workforce. Surgical trainees are prepared for the mentored research experience far in advance of the T32 training period in order to optimize their research success and productivity. Each trainee dedicates a minimum of two years without clinical responsibilities to conduct mentored research, participate in immunology focused didactics (earning a Masters of Medical Science (MMS) degree) and engage in professional development activities. MMS requirements include coursework in Immunology, Rigorous Research Design, Biostatistics, Grant Writing, and Research Ethics. Trainees are required to take “Career Development for Surgeons” course and “Applications of Immunology in Surgery” journal club. Trainees also have ample opportunity to participate in OSU institutional research workshops, seminars and career development activities. After completion of the research training program, trainees transition back to complete 2-3 more years of surgical training. During this time, they continue to publish, present at national meetings, compete for awards and participate in research and professional development activities. To date, 17 trainees (4 URM) have been appointed, 2 are in training, 15 completed the program (no attrition), achieved high productivity (publications, presentations, competitive awards), 3 exceeded program milestones and earned PhD degree. Ten of 15 who completed both research and clinical training placed into prestigious fellowships (9) and one proceeded directly into an academic faculty position and 5 remain in clinical training. We plan to appoint 5 postdoctoral trainees per year, each for 2 years duration.