University Of Rochester

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract The prefrontal cortex (PFC) is widely reported to play a role in visual attention, employed through modulation of neuronal activity in visual processing regions, such as V4 and MT. Sub-regions within the PFC of monkeys, such as the frontal eye fields (FEF) and the ventral prearcuate region (VPA), are reported to govern spatial and feature-based attentional modulation of visual responses, respectively. However, the cellular-level circuitry and neurocomputational mechanisms involved in the modulatory control of visual processing remain unclear. Previous work suggests that interactions between diverse sub-populations in both FEF and VPA may facilitate modulation of visual processing signals as well as shifts of attention, but this remains untested. The purpose of my project is to determine the neuronal dynamics within sub-regions in PFC that govern top- down modulation of visual processing. It is possible, that FEF may integrate signals representing object identity which arise in VPA and transforms them to signals representing spatial locations of a target stimulus. We plan on addressing this hypothesis by: (1) characterizing the spatial- and feature-tuning properties of FEF in the context of visual search to understand how integration and transformation of attentional signals may be implemented; (2) establishing the temporal dynamics between FEF and VPA to reveal the nature of neuronal communication between both regions during visual search; and (3) determine the causal role of VPA towards feature-based attentional modulation of visual processing via reversible pharmacologic inactivation. Monkeys are trained on a visual search task designed to conditionally engage attentional processes that are dependent on feature or spatial information. Using neurophysiology and population analyses, we will determine the neuronal correlates of PFC which govern the computational processes needed to accomplish these tasks. These findings will provide a description of the circuit-level mechanisms involved in top-down attention which often times is affected in neuropsychological disorders and neurodegenerative diseases. Thus, this project aims to contribute knowledge concerning the neuronal correlates of visual processing that are involved in related underlying symptoms. Additionally, learning about how higher-level brain regions modulate visual processing may provide more information about how perception is conjured in the brain. My environment at UR fosters the opportunity to broaden my research skillset which ultimately facilitates my endeavor towards my career goal as a scientific leader pushing to improve our approach of intervening with mental illnesses. The social environment at UR provides great opportunity for outreach, allowing me to fulfill my aspirations as a community leader looking to pave a way for underrepresented individuals which get overlooked given their backgrounds.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Human immunodeficiency virus (HIV-1) infection can lead to the deadly disease acquired immunodeficiency syndrome (AIDS). During the natural infection of HIV-1, some viral DNAs are integrated into host genome, but the vast majority of viral DNAs exist in an unintegrated state. Transcriptional regulation of unintegrated HIV-1 DNA plays important roles in HIV-1 infection and pathogenesis. In contrast to the robust viral gene expression from integrated viral DNA, the extrachromosomal, unintegrated viral DNAs are very poorly transcribed. The exact mechanisms for the silencing of unintegrated HIV-1 DNA are not well understood, which constitutes a major knowledge gap in HIV-1 research. HIV-1 accessory protein Vpr enhances viral gene expression from unintegrated HIV-1 DNA by targeting host proteins for degradation. In search for Vpr target host factor(s) that can silence unintegrated HIV-1 DNA, we have performed a CRISPR-Cas9 knockout screening of Vpr target genes and identified NS1BP. We also found that NS1BP-interacting partner, the SMC1A/3 cohesin complex, is required for the silencing of unintegrated HIV-1 DNA. We hypothesize that NS1BP acts as a cofactor to facilitate the loading of the SMC1A/3 cohesin complex on viral DNA, which results in viral chromatin compaction and gene suppression, and Vpr-mediated degradation of NS1BP results in the dissociation of the cohesin complex from viral DNA, which consequently depresses the silencing of unintegrated HIV-1 DNA. In this project, we will determine the mechanism by which NS1BP and the SMC1A/3 cohesin complex mediate the silencing of unintegrated HIV-1 DNA (Aim 1), and elucidate the mechanism by which Vpr antagonizes the silencing mediated by NS1BP and the cohesin complex (Aim 2). Our proposed studies will significantly extend our understanding of the molecular mechanism for the transcriptional regulation of unintegrated HIV-1 DNA and provide new information regarding the epigenetic silencing of HIV-1 DNA. In the long term, these studies will provide new targets and strategies for the cure of HIV-1 infection.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Military suicide rates increased 61% from 2008 – 2019 and rates have increased faster in the U.S. Air Force (USAF) compared to other branches. Currently, the predominant military suicide prevention approach is to try to remediate suicide risk after suicidal individuals are identified. No RCT-validated universal programs shown to reduce vulnerability to suicide are in wide use. To fill this gap, the Wingman-Connect Program is a group-based intervention that strengthens protective relationship networks and skills for managing career and personal challenges, to reduce vulnerability to suicide across the broad USAF population. This proposed Hybrid Effectiveness-Implementation trial tests the effectiveness of the Wingman-Connect Program on individual suicide risk and on base-level suicide attempts. We will examine theory-driven mediators and moderators and implementation of the program as delivered by US Air Force (not research) personnel under real world conditions across 2 sequential early career phases. This effectiveness study is the critical next stage in the translational pipeline toward large-scale roll-out to prevent suicide deaths. To rigorously test effectiveness, we co-developed with USAF partners a 2-stage randomized design. (1) The first stage of randomization will be at Initial Technical Training, in which 396 classes of USAF personnel will be randomized to Wingman-Connect or to an active control (N=2,970 Airmen) and followed for one year. These classes send a high proportion of graduates to 8 operational bases. (2) The second stage of randomization will occur among these 8 operational bases, which will be randomized in pairs to start implementing WC at 4- month intervals (stepped wedge design). Once WC has been initiated at each base, all entering first-term Airmen will receive WC, with ~17,400 total Airmen trained across all bases. This 2-stage design will yield robust, multi-level effectiveness findings. Aim 1: Test effectiveness of WC on reducing (a) self-reported suicide risk and (b) base-level rates of suicide attempts. We will evaluate (a) individual level outcomes of suicide risk, depression, and occupational problems up to 1 yr; and (b) base-level administrative records of suicide attempts. Aim 2: Evaluate theory-proposed network health mediators and moderators. WC is expected to increase Airmen's positive social bonds, group cohesion, morale, and healthy coping norms in their social networks; those changes will contribute to reduced suicide risk, depression and occupational problems. Aim 3: Examine implementation determinants and mechanisms, and refine Implementation Package. Key implementation outcomes will be USAF implementers' fidelity delivering WC (n=24-30) and engagement in training/technical support. Implementer fidelity and engagement is expected to be predicted by base implementation climate and WC embeddedness into base communications and support activities.

NIH Research Projects · FY 2024 · 2023-08

Project Summary and Abstract This award will support Dr. Jensen-Battaglia’s long-term goal of developing the expertise and skills needed to become an independent investigator exploring the overlapping roles of physical function and environment in health outcomes for older adults with cancer. By 2050, 20% of new cancer diagnoses will be among those age 80 or older, comprising an estimated 6.9 million cases worldwide. The majority of older adults in the United States (U.S.) prefer to remain in their homes as they age (i.e., ‘age in place’), which is associated with health benefits. Conversely, both unsupportive neighborhood environment and residential relocation are associated with increased healthcare utilization, decreased survival, and falls. Although impairments in physical function are common and highly modifiable, we do not yet know if older adults with cancer are at greater risk for residential relocation as a result of these impairments compared to those without cancer. Whether negative health outcomes such as healthcare utilization, mortality, and falls associated with relocation are due to relocation itself or associated changes in the neighborhood environment remains unclear. Dr. Jensen-Battaglia will address these gaps in her proposed project by prospectively evaluating the association between impairments in physical function and residential relocation among community dwelling older adults in the U.S., and examining how this association differs for those with cancer compared to those without cancer. Additionally, she will assess how residential relocation modifies the effect of neighborhood environment on future health for those with cancer. For the F99 phase, Dr. Jensen-Battaglia will leverage data from a nationally representative sample of U.S. Medicare beneficiaries [National Health and Aging Trends Study (NHATS)] to refine a novel measure of mobility-related physical function, assess whether this is associated with increased risk of residential relocation, and evaluate if cancer diagnosis positively modifies this relationship. She will also explore how the relationship between mobility-related physical function and residential relocation differs across home environments and cancer types. For the K00 phase, she will create a dataset linking the NHATS with Medicare claims and area-based measures, and evaluate if residential relocation positively modifies the effect of worsening (compared to stable or improving) neighborhood environment supports on cancer-related health outcomes. Dr. Jensen-Battaglia has worked closely with her sponsors to develop a training plan which supports successful completion of the predoctoral research project and smooth transition to a competitive, cancer-focused postdoctoral placement. This includes training to improve knowledge of the patient cancer care experience, obtain expertise in analysis of residential relocation dynamics, and identify a postdoctoral mentor. Together, the proposed research and training plan provide optimal opportunities and structure for Dr. Jensen- Battaglia to develop new skills and progress toward becoming an independent cancer researcher.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Dexterous manipulation of objects relies on brain computations that integrate neural signals encoding tactile signals on the skin with the proprioceptive state of the hand. In particular, neural ensembles that encode tactile motion on the skin are critical because they provide feedback signals to motor planning areas to indicate whether an object is slipping from the hand. Although we have a good understanding of how tactile motion signals are represented in the brain, this knowledge has been accrued from studies that placed the hand in a fixed position. Indeed, all studies that have investigated tactile motion mechanisms at the single-cell level have done so in animals not performing a motion discrimination task, and with their hands placed in a fixed posture. These studies show that tactile motion can be represented in area 1 by cells that integrate different tactile cues of the object (e.g., direction, speed, and saliency). However, our recent work in humans shows that perception of tactile motion on a finger is modulated by the proprioceptive state of the hand, and the body part location in which motion judgements are made relative to (i.e., the reference frame). These findings indicate that current models of tactile motion require major revisions. That is, neural models of tactile motion should take into account how motion representations in touch are transformed by proprioception and/or reference frame signals. Thus, the overarching goal of this application is to determine the neural areas and mechanisms that generate reference frame-specific representations of tactile motion. Key to determining the single-cell mechanisms that mediate reference frame-specific representations of tactile motion is to record activity in non-human primates (NHPs) discriminating tactile motion stimuli in different reference frames, and with their hands placed in different postures. Unfortunately, our field does not have an established paradigm, or training regime, to study these motion mechanisms in a NHP. In Aim I, we develop a behavioral paradigm to train NHPs to discriminate the motion direction of stimulus on a finger (e.g., index finger) in different reference frames (e.g., relative to the center of the body, or the thumb), while their hands are placed different proprioceptive states (e.g., pronated vs. supinated). In Aim II, we will record single-unit activity in somatosensory cortex (area 1) in trained NHPs to determine the neural computations that generate reference frame-specific representations of tactile motion. Our behavioral experiments will test that perceptual representations of tactile motion are conserved across NHPs and humans, demonstrating that NHPs are a viable species to study neural mechanisms of tactile motion at the single cell level. Our neurophysiology experiments will test whether motion selective neurons in area 1, the tactile analogue of medial temporal (MT) cortex for visual motion, flexibly represent motion in different reference frames. This project will demonstrate, for the first time, behavioral and neurophysiological evidence of NHPs performing tactile motion discrimination tasks, a crucial prerequisite for a competitive R01-type application.

NIH Research Projects · FY 2026 · 2023-08

Abstract Autograft is considered the gold standard for large bone defect repair and reconstruction. The superior healing potential of autografts is attributed to the robust osteogenic and angiogenic activities of periosteum– a highly vascularized thin tissue membrane covering the outer surface of bone. To recapitulate the superior healing potential of periosteum, a series of tissue engineering strategies have been developed, aiming to construct a biomimetic tissue-engineered periosteum (TEP) for enhanced bone defect repair and reconstruction. The success of these strategies hinges on a thorough understanding of the osteogenic and angiogenic role of periosteum and a better insight into the intricate relationship between bone and vessel forming cells at the regenerative interface of periosteum-mediated repair. Supported by NIH funding, we have made a series of progresses in construction of a biomimetic functional periosteum and in understanding of the functional vascular bed at the site of periosteal repair utilizing transgenic animals that label subtypes of endothelial cells. Building on these progresses, the proposed work will focus on understanding the molecular control of periosteum-mediated bone-specialized vessel formation, with further efforts devoted to building a technological platform for controlled delivery of angiogenic and osteogenic factors for reconstruction of a pro- angiogenic and pro-osteogenic periosteum mimetic for augmented bone allograft repair. The completion of the proposed study will provide new insights into the molecular control of bone-specialized blood vessel formation during periosteum-mediated repair, further offering engineering-based strategies targeting osteogenic and angiogenic interface for augmented defect repair at a compromised periosteal site.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The development of general methods for the construction of sp3-rich (i.e. highly three-dimensional) organic scaffolds is a longstanding challenge in organic synthesis. High sp3 character imparts beneficial biological activities and pharmacokinetic properties into organic molecules, but because of their complexity, such compounds are underrepresented in libraries for drug discovery relative to sp2-rich compounds. Transition metal catalysis has revolutionized the construction of sp2-rich organic scaffolds, producing an array of general transformations that allow for the facile synthesis of diverse libraries of compound analogues for developing novel small molecule therapeutics. To establish similarly versatile methods for synthesizing sp3-rich organic scaffolds from simple starting materials, innovations in catalysis are needed. The research proposed herein employs innovative ligand and catalyst design as a means to discover novel and general scaffold-building methodologies that can transform simple starting materials (i.e. alkenes, dienes, arenes) into functionally and structurally complex products. In one area, we are developing unconventional ligand platforms that occupy underpopulated regions of ligand space for Pd catalysis for the development of olefin carbofunctionalization reactions. We have found that ligands derived from urea, which occupy a region of small organic ligands that is inaccessible to phosphines and N-heterocyclic carbenes, effectively promote heteroannulation reactions of ambiphiles and dienes. In addition, phosphine ligands with unconventional steric profiles can exert ligand control over site-selectivity for heteroannulations with dienes. Future work in this area will focus on expanding on these findings to develop a unified synthetic approach to preparing diverse aliphatic heterocycles, as well as selective, multicomponent carbofunctionalization reactions of olefins. These methodological developments will be enabled by both rational ligand design and computationally-aided ligand discovery. In another area, we are establishing Cu-diamine complexes as general catalysts for oxidative, radical addition reactions. Central to our reaction design is coordinating Cu catalysts to the substrate, which promotes selective generation of reactive radical intermediates that can add to olefins and arenes. Using this approach, we have discovered an aerobic amino- oxygenation of internal alkenes that engages diverse aryl-substituted alkenes and operates under mild conditions. Designing ligands that enhance the oxidative potential of Cu and facilitate coordination to substrates will enable the discovery of new catalytic reactivity in oxidative, radical olefin addition reactions, and provides a framework for the development of highly enantioselective transformations. These reactions will enable the rapid construction of diverse functional motifs and cyclic scaffolds with excellent catalyst control over chemoselectivity and stereoselectivity. In total, the proposed research program will result in the development of versatile catalytic methods for the efficient preparation of functional molecules that are relevant for discovering compounds with therapeutic potential, and thus will have a significant impact on biomedical sciences and human health.

NIH Research Projects · FY 2025 · 2023-08

This MIRA application extends our decades-long research on nonsense-mediated mRNA decay (NMD) and how NMD factors can function in other aspects of cellular metabolism. NMD is a fundamental biological process by which mammalian cells eliminate mRNAs containing a nonsense codon deriving from a genetic or acquired frameshift or nonsense mutation. NMD also eliminates an estimated one-third of mRNAs that cells produce by routine mistakes made during gene transcription and/or mRNA production. Over the years, we have worked to elucidate the molecular mechanism of NMD. As one of many outcomes, we have established a “rule” that clinicians and researchers use to predict which nonsense codons result in recessively inherited vs. dominantly inherited disease. We have also demonstrated how cells regulate the efficiency of NMD as an adaptive mechanism during changing environments, e.g. during development, differentiation, or drug treatments. This application pursues our serendipitous finding that NMD is hyperactivated in fragile X syndrome (FXS), which is the most common single-gene cause of intellectual disability and autism, affecting 1/4000 boys and 1/6000-8000 girls. We aim to understand how the protein that is missing in FXS functions via interactions with other proteins and mRNAs to protect these mRNAs from translation and decay. We also aim to decipher the mechanism by which the RNA-binding protein Staufen prevents a runaway immune response. On another front, our long-time interest in mechanistic connections that span pre-mRNA splicing in the nucleus to mRNA translation and decay in the cytoplasm will be extended to include gene transcription and nuclear mRNA decay. We have long been fascinated by the structural dynamics and functions of the largely nuclear cap-binding heterodimer CBP80−CBP20, which binds co-transcriptionally to the 5'-cap of nascent pre-mRNAs. While our past interests have focused on the role of CBP80−CBP20 in the pioneer round(s) of cytoplasmic translation, during which we have shown exon-junction complex-mediated NMD occurs, we aim to understand roles of CBP80−CBP20 in the nucleus. As one example, we are studying the mechanism by which a master transcriptional co-activator of genes whose products regulate critical cellular processes engages with CBP80−CBP20 so as to promote the expression of an understudied category of RNA polymerase III- transcribed genes. In related work, we are studying connections between CBP80−CBP20 and the little- understood, and so-called, nuclear cap-binding protein (NCBP)3. We aim to elucidate the significance of our finding that NCBP3 regulates newly made mRNAs from genes encoding proteins that function in mitochondrial biology. These connections will be examined in skeletal-muscle cells in vitro and ex vivo, the latter using mice, which should lend insight into the etiology and pathogenesis of many human diseases that include sarcopenia, neuromuscular disorders, and cardiomyopathies. While our interests are broad, they are connected by the goal to understand molecular mechanisms in health and in disease, with a focus on RNA metabolism.

NIH Research Projects · FY 2025 · 2023-08

Heart disease is the leading cause of morbidity and mortality worldwide. In healthy myocardium, the mitochondria utilize oxidative phosphorylation to generate ATP and metabolites to support pumping blood throughout the whole body. Given this critical function of mitochondria in the heart, mutations or reductions of essential mitochondrial factors cause mitochondrial cardiomyopathy (MC) in humans and mice. Mitochondrial dysfunction is also a major pathogenic driver in non-genetic ischemic heart disease such as myocardial infarction (MI). Better understanding of mitochondrial protein functions and pathogenic molecular mechanisms underlying mitochondrial dysfunction will promote the development of therapeutics for MC or MI. Recent RNA-seq coupled with ribosome footprint-seq analyses in mouse hearts reveal Fam210a (family with sequence similarity 210 member A) as a hub gene in cardiac remodeling. Our preliminary data suggests reduced FAM210A expression in mouse MI hearts and human ischemic heart failure. Cardiomyocyte (CM)-specific homozygous (Homo) conditional knockout (cKO) of Fam210a in adult mice led to MC and mortality. Interactome analyses reveal that FAM210A binds to mitochondrial Ca2+/H+ exchanger LETM1 (Leucine zipper and EF-hand containing transmembrane protein 1) and promotes mitochondrial Ca2+ (mCa2+) efflux in vitro and in vivo. Therefore, Fam210a deletion in CMs resulted in an elevated mCa2+ and reactive oxygen species and compromised mitochondrial membrane potential. As a result, the mitochondrial respiratory activity was reduced in Fam210a KO CMs, leading to cardiac dysfunction at a late stage. In addition, persistently activated integrated stress response (ISR) contributed to the disease progression in Fam210a cKO hearts. Moreover, CM-specific heterozygous Fam210a cKO mice exhibited lower FAM210A protein expression and more severe cardiac remodeling than control mice under MI. In contrast, AAV9-mediated overexpression of FAM210A could protect hearts from MI-induced cardiac damage and dysfunction. Our central hypothesis is: FAM210A functions as a mitochondrial Ca2+/H+ antiporter regulator and maintains normal mitochondrial and cardiac function. We will test this hypothesis by pursuing 3 aims. Aim 1. Decipher the molecular mechanism of FAM210A in regulating mCa2+ homeostasis. Aim 2. Elucidate the role of FAM210A in regulating cardiac mitochondrial activity and cardiac function. Aim 3. Determine the effects of FAM210A overexpression on the functional performance of mitochondria, CMs, and the heart under MI. Collectively, our studies provide novel insights into the function and mechanisms of FAM210A in regulating cardiac mitochondrial integrity and thus maintaining the normal physiological function of the heart. This project also suggests that reduced FAM210A level contributes to the MI-induced cardiac pathological remodeling and overexpression of FAM210A has a cardioprotective role in MI treatment.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT Transition metal-catalyzed cross-coupling methods are important in the context of human health because they play a central role in the synthesis of small-molecule therapeutics and molecular probes. Although cross-coupling methodologies are dominated by palladium catalysis, newer methodologies utilizing terrestrially abundant 3d metals (such as nickel) enable cross-coupling with polar C–O and C–N electrophiles. This feature is important because O- and N-containing functional groups are common in bioderived and bioactive small molecules and could therefore offer a greatly expanded scope of sustainably sourced cross-coupling partners. However, nickel catalyzed methodologies for cross-coupling with acyl C–O and C–N electrophiles remain in early stages, and (i) face practical limitations due to a pronounced sensitivity to changes in substrate structure, (ii) generally require high precatalyst loadings, and (iii) often utilize high reaction temperatures or long reaction times. Attempts to address these limitations are stymied by a lack of detailed mechanistic into the features responsible. This proposal addresses these ambiguities through systematic mechanistic investigation of three distinct classes of nickel-catalyzed cross-coupling with biologically important acyl C–O and C–N electrophiles with a specific focus on (i) C–X activation steps, (ii) selectivity-determining features, and (iii) speciation of key organometallic intermediates. This approach leverages ligand design and organometallic synthesis, structure elucidation through spectroscopic and crystallographic studies, and reaction kinetics, supported by state-of-the-art computational analysis to derive insights into the reactivity and selectivity-determining features of catalytic reactions. These insights will be leveraged to elucidate key reactivity and selectivity relationships and to offer methodological improvements that address current inefficiencies. As an Early-Stage Investigator (ESI), the PI is uniquely suited to build this research program due to their extensive prior experience working across the organic– inorganic and synthetic–mechanistic axes to interrogate, improve, and invent methodologies with translational potential. The PI’s program will build on this expertise to offer conceptually innovative, mechanism-driven, strategies to meet key synthetic needs. Successful completion of the proposed research will result in detailed insight into the mechanisms and limiting features of nickel-catalyzed acyl C–N and C–O activation and cross coupling. These insights will be translated into development of a suite of single-component precatalysts with enhanced activity and selectivity along with a practical “user’s guide to catalyst selection” to enable expanded application to the synthesis and elaboration of biologically important small molecules. The ultimate goal of this project area is to achieve mechanism-driven improvements bringing these methodologies—which use terrestrially abundant metals to elaborate biologically abundant functional groups—to a level competitive with or superior to established, palladium-catalyzed cross-coupling alternatives.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Chromosomal translocations involving Nucleoporin 98 (NUP98) are observed in approximately 5% of pediatric acute myeloid leukemia (AML) and are associated with resistance to therapy and poor outcome, with approximately 35% 5 year overall survival. NUP98 rearrangements lead to expression of oncogenic chimeric gene fusions involving the intrinsically disordered, N-terminal region of NUP98 and the C-terminal region of one of over 30 identified partner genes. The partner genes commonly have domains with key functional properties, including homeodomain moieties (e.g. HOXA9) and roles in transcriptional regulation (e.g. NSD1, KDM5A). In complex with other machinery needed for gene regulation, NUP98 fusion oncoproteins (FOs) bind to the promoters of many developmental genes. This leads to changes in chromatin structure, increased expression of target genes, and aberrant hematopoietic self-renewal. Recent studies, including my own, have shown that the ability of NUP98 FOs to localize within the nucleus in membrane-less organelles, or “puncta” formed through liquid-liquid phase separation (LLPS), is necessary for transformation and deregulated gene expression phenotypes. Nevertheless, which proteins interact with NUP98 FOs in puncta and the importance of puncta formation for effective therapeutic targeting of NUP98-rearranged cells is not known. This research proposal seeks to identify the proteins found in NUP98 FO-associated puncta, uncover how puncta alter gene regulation, and determine if puncta disruption correlates with effective treatment of NUP98-rearranged cells. Aim 1 will examine the role of histone acetyltransferase (HAT) complex members, which my preliminary data identified as key NUP98 FO interacting proteins, in NUP98::KDM5A FO-driven cell transformation. I will perform CRISPR/Cas9 editing of HAT complex genes in hematopoietic stem and progenitor cells (HSPCs) from our Nup98::Kdm5a mouse model and study the in vitro and in vivo consequences of these alterations. I will also examine gene expression and chromatin remodeling in Nup98::Kdm5a HSPCs with and without HAT complex disruption. Aim 2 will determine whether effective therapeutic targeting of NUP98 FOs leads to puncta disruption. I will perform co-localization experiments for FO with proteins involved in nuclear transport and gene regulation. I will then pharmacologically inhibit these interacting proteins using available small molecule inhibitors and assess changes in puncta features and cell viability over time to determine if puncta disruption correlates with drug efficacy. In Aim 3, I will identify interacting proteins that are vulnerabilities in NUP98-rearranged cells and use pharmacologic inhibition of crucial interactors to identify how they are involved in cell transformation, gene regulation, and LLPS. Together, these studies will identify critical interacting proteins in leukemias bearing NUP98 gene fusions, examine how they contribute to leukemogenesis, and uncover how they might be targeted for therapeutic benefit.

NIH Research Projects · FY 2025 · 2023-08

Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure residing in the cardiac intercalated disc (ID). Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC), a disease characterized by high propensity to life- threatening arrhythmias and myocardial structural damage, often of right ventricular predominance. Much attention has been given to the loss of cell-cell attachment at the ID as a disease mechanism. Yet, it is becoming evident that PKP2 mutations also lead to an array of poorly understood cardiomyocyte (CM)-intrinsic disturbances. Desmin intermediate filaments are anchored to the desmosome in a PKP2-dependent manner, supporting CM structural integrity and facilitating communication from the cell surface to the nucleus. Our prior work in mouse models and human patient samples found PKP2 mutation disrupts CM nuclear envelope (NE) integrity and leads to DNA damage. Based on published reports and our preliminary data, we hypothesize that PKP2 deficiency, or disease relevant PKP2 mutations, disrupt the structural, functional and molecular integrity of the cardiomyocyte nuclear envelope, leading to genomic reorganization, the DNA damage response, and altered transcription. The following aims will investigate how PKP2 deficiency disrupts the nucleus to accelerate ARVC disease progression. Aim 1: Define the impact of PKP2 deficiency on the cardiomyocyte nuclear lamina protein interactome. We hypothesize that PKP2 deficiency alters the proteome of the cardiomyocyte NE, and that this disruption is an early trigger for the disease phenotype. We will interrogate changes in the molecular ecosystem of the cardiomyocyte NE after loss of PKP2 expression using proteomics and single molecule imaging. Aim 2: Define the impact of PKP2 deficiency on cardiomyocyte genomic organization. We hypothesize that loss of NE integrity in PKP2 deficient CMs disrupts genomic organization at Lamin Associated Domains and causes transcriptional remodeling. We will determine how structural damage is transmitted from the cell membrane to the genome, focusing on changes that occur in the vicinity of the NE through advanced imaging, genomic and transcriptomic approaches. Aim 3: Investigate strategies to reduce DDR and delay cardiomyopathy in PKP2 deficient mice. We hypothesize data that PKP2 mutation induces P53-dependent DNA damage response (DDR), which may exacerbate ARVC disease progression. Genetic epistasis experiments and pharmacological approaches will investigate how the P53-dependent DDR contributes to PKP2-dependent cardiomyopathy. Defining pathological changes to nuclear architecture that precede overt myocardial structural remodeling will reveal exciting opportunities for new therapeutic strategies aimed at slowing ARVC disease progression by restoring nuclear envelope homeostasis or preventing the DNA damage response.

NIH Research Projects · FY 2026 · 2023-08

Computations performed by single neurons result from integration of a large myriad of synaptic inputs distributed throughout complex dendritic topology. Synaptic inputs vary in substantial ways; sensory-driven activity patterns, probability of activation, synapse location within the dendritic topology, local dendritic organization, and ultrastructural characteristics are all thought to be critical for determining how synaptic inputs influence the spiking output of a neuron. Populations of synaptic inputs, ultimately, determine the coding capacity and computations single neurons can perform. Despite this fact, studies largely overlook the synaptic input population within a single neuron, instead focusing on the activity of cellular populations, using biophysical models or constructing models that create hypothetical weights or synapses (e.g. deep-neural networks). Thus, a critical question in neuroscience remains how ensemble synaptic activity is integrated in vivo and what are the fundamental principles of synaptic organization which describe neural computation. To address this issue, we present a tightly integrated experiment-theory approach. We propose to (1) measure the sensory-driven activity patterns of large populations of dendritic spines on layer 2/3 visual cortical neurons in ferret visual cortex in vivo, and (2) use a statistical physics approach to characterize the structure and computing of synaptic populations in multiple contexts. Thus, this project will provide fundamental knowledge about the synaptic architecture of neurons in the brain. The

NIH Research Projects · FY 2025 · 2023-07

Project Summary Eukaryotic genomes contain a diversity of evolutionarily “selfish” genetic elements (SGEs) that obtain transmission advantages at the expense of their host carriers. SGEs tend to use one of two broad strategies: over-replicate relative to the host genome (e.g., transposable elements) or distort fair Mendelian transmission (e.g., meiotic drive elements). Sex chromosomes are especially susceptible to the invasion and accumulation of meiotic drive elements that, by distorting X versus Y chromosome transmission via selective sperm-killing, bias progeny sex ratios. Such “sex-ratio drive” can potentiate evolutionary conflicts of interest between drive genes and drive-suppressors at X-linked, Y-linked, and autosomal loci. Molecular arms races arising from recurrent bouts of such drive-mediated conflict can have far reaching consequences for male fertility, for demography (including the potential risk of unisexual population extinction), for genome evolution, and for speciation. Understanding the genetics, molecular mechanisms, evolutionary dynamics, and genomic consequences of sex-ratio drive is therefore of paramount importance. Here we propose to investigate a system of sex-ratio drive and suppression that evolved recently and then, as expected under molecular arms race dynamics, rapidly diversified among three closely related species of Drosophila— D. simulans, D. mauritiana, and D. sechellia. Our previous work revealed that, while these species diverged ~250 Kya and are today reproductively isolated by numerous genetic incompatibilities, some gene flow has nevertheless occurred among all three. We discovered that the so-called Winters sex- ratio drive system is part of a family of X-linked, satellite DNA-associated, Dox-like (Dxl) drive genes that amplified in copy number and diversified in sequence. These Dxl genes encode predicted DNA-binding sperm-specific histones, which we predict differentially disrupt chromatin remodeling of X- versus Y-bearing spermatid pronuclei during spermiogenesis. Furthermore, we found that each species possesses two hairpin RNA-producing autosomal suppressor genes that produce endogenous small interfering RNAs (esiRNAs) predicted to specialize in the silencing of different subsets of the Dxl gene family. Finally, we discovered that some Dxl genes and suppressors show population genetic signatures of strong recent selection and interspecies gene flow. Our research project aims to combine experimental genetics, molecular biology, third-generation sequencing, and evolutionary genomics methods to dissect Dxl driver- hpRNA suppressor interactions; to determine the genomic binding targets of the Dxl-encoded histones and its implications for the evolution of Y chromosome sequence content; and to infer the evolutionary history and dynamics of Dxl-mediated molecular arms races, including its implications for the evolution of hybrid sterility and interspecific gene flow.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Speech is central to human life. Yet how the human brain converts patterns of acoustic speech energy into meaning remains unclear. This is particularly true for natural, continuous speech, which requires us to efficiently parse and process speech at multiple timescales in the context of our ongoing conversation and situational knowledge. Much progress has been made on this problem in recent years by the realization that the dynamics of cortical activity track those of natural speech. This has led to the development of new methods to study the neurophysiology of speech processing in more naturalistic paradigms. However, the field still lacks consensus regarding the precise physiological mechanisms and neurostructural origins of this tracking. In particular, two contrasting theories have been advanced that attempt to explain the genesis of this phenomenon. The first proposes that the quasi-rhythmic nature of continuous speech “entrains” intrinsic, endogenous oscillations in the brain as a way to parse that continuous speech into smaller units for further (linguistic) processing. Meanwhile, the second proposes that the cortical tracking of speech reflects the summation of a series of transient evoked responses from hierarchically organized neural networks that are tuned to the different acoustic and linguistic features of speech. The contrast between these two ideas is reflected in the emergence of two almost completely non-overlapping literatures in the field of speech electrophysiology. This is highly problematic as the design and interpretation of most studies on this important topic are now filtered through either one or the other of these theoretical lenses. Without a clear understanding of the true mechanisms involved, our collective work on this topic thus runs the risk of being distorted through misconception. This project aims to address this urgent need by critically examining these two frameworks side by side. We aim to do so by collecting scalp EEG from human adults as they listen to natural and manipulated speech. These manipulations will involve varying speech across several dimensions that should maximize the differences in the predictions made by each theory. We specifically aim to test the hypothesis that both evoked responses and entrained oscillations contribute to the cortical tracking of speech, with their relative contributions varying as a function of the statistics of the speech and attention. We will test this hypothesis by analyzing the EEG data with reference to computational models of both evoked and oscillatory activity. Furthermore, we will use the same analytical framework to model signals from different regions of the speech/language processing hierarchy acquired using intracranial recordings in neurosurgical patients. This will allow us to test the deeper hypothesis that evoked and oscillatory mechanisms operate differently in different cortical areas. We will also leverage these intracranial findings to develop a fuller understanding of our scalp EEG signals with a view to strengthening the applicability of our work to future cognitive and clinical research.

NIH Research Projects · FY 2025 · 2023-07

The goal of this project is to adapt the empirically-validated FOCUS intervention and test its feasibility and acceptability among high-risk cancer patients and their caregivers. High-risk cancer patients face unaddressed challenges that adversely impact their quality of life (QOL), including chronic stress caused by living within specific high-risk communities. Furthermore, caregivers of high-risk patients are often not acknowledged in healthcare systems primarily designed to serve patients in general; resources for caregivers of patients in general often do not address their needs; high-risk patients may be more likely to rely on friends or ex-partners as caregivers; and these caregivers may be affected by chronic stress in cancer care settings. Psychoeducational interventions are needed to address these challenges and improve the QOL of the cancer patients and their caregivers together. The FOCUS intervention uses both psychoeducation and problem solving to improve QOL, coping, and self-efficacy in cancer patients and their caregivers, and has been shown to be efficacious in three prior clinical trials. However, as noted by our community partners, the Cancer Action Council (CAC), content has not been tailored to high-risk populations. Consequently, we propose to adapt the evidence-based FOCUS Program for high-risk cancer patients and their caregivers (Aim 1). In collaboration with the CAC and guided by a theoretical adaptation model and ADAPT-ITT framework, we will conduct 6 focus groups with ≥24 high-risk cancer patients and their ≥24 caregivers to “theater test” FOCUS and elicit qualitative feedback about adaptation. We will also elicit feedback from our panel of 5 topic experts in cancer research, and create a manualized, adapted intervention called FOCUS-HR. We will then assess the feasibility and acceptability and explore potential outcomes of the adapted FOCUS-HR intervention in a two-arm pilot trial (Aim 2). We will randomize 80 high-risk cancer patients and their caregivers (total N≥160) to either FOCUS-HR or a waitlist control. Assessment of patients and caregivers will occur at baseline, post-intervention, and at a 3-month follow-up (~6 months after baseline). We hypothesize that it will be feasible to recruit a sample of high-risk cancer patients and caregivers through clinic, community, and online approaches; at least 80% of high-risk patients and caregivers will be highly satisfied with the content and delivery of FOCUS-HR; 75% will complete 3 or more FOCUS-HR sessions; and 75% will complete post-intervention and 3-month follow-up assessments. We will also explore whether potential outcome measures (QOL, coping, self-efficacy to manage cancer, relationship communication, and stress) are responsive to the effect of FOCUS-HR versus control. This grant represents a critical step in preparing our team to conduct a definitive trial of FOCUS-HR, responds to community and NCI priorities, and has potential to address QOL in high-risk populations.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT Gestational diabetes is characterized by chronic maternal hyperglycemia during pregnancy without a prior diagnosis of diabetes. It is a very common obstetric complication affecting ~10-25% pregnant women globally. Because women with gestational diabetes are more likely to have other pregnancy complications, deliver large for gestational or premature babies, and develop type II diabetes, gestational diabetes poses a serious threat to the health of mother and baby. Although some risk factors have been defined, the underlying mechanisms are complex and the precise etiologies are poorly understood. Recent studies show that pancreatic serotonin signaling plays a critical role in maternal glucose homeostasis. Increased serotonin synthesis in the pancreatic islet is a critical event that promotes beta cell proliferation and increased insulin secretion that are needed to prevent maternal hyperglycemia during pregnancy. Dietary and genetic factors that reduce islet serotonin synthesis are causatively linked to gestational diabetes in mice. Our preliminary studies show that low dose exposure to perfluorooctanoic acid (PFOA) is associated with reduced abundance of serotonin and its critical cofactor vitamin B6 in the pancreas from pregnant C57BL/6 mouse. These results are consistent with epidemiological findings that PFOA exposure in pregnant women is linked to maternal hyperglycemia, insulin resistance, and glucose intolerance. Interestingly, DBA/2J mice exposed to PFOA do not develop gestational diabetes. The C57BL/6 and DBA/2J mice differ in their abilities to metabolize vitamin B6 due to differences in activities of alkaline phosphatase (ALP). These results suggest that environmental exposure-induced gestational diabetes is modulated by genetic background and higher endogenous vitamin B6 level confers a protection. The overall hypothesis is that PFOA exposure in pregnant mice is causatively linked to gestational diabetes through mechanisms that perturb serotonin metabolism in maternal pancreatic islets and the effects are modulated by genetic differences in vitamin B6 bioavailability. We propose to investigate beta cell proliferation and serotonin abundance in control and PFOA-exposed pregnant C57BL/6 mice to determine whether the gestational diabetes is causatively linked to reduced beta cell expansion and reduced insulin secretion. We also wish to investigate whether treatment with an ALP inhibitor in the DBA/2J pregnant mice will reduce vitamin B6 in pancreatic islets and result in loss of protection to gestational diabetes. Finally, to determine how pregnancy and gestational diabetes influence islet programming, we will perform RNA sequencing and Cleavage Under Targets and Tagmentation followed by sequencing to study changes in the transcriptome and epigenome in response to physiological changes and disease. The proposed research will provide knowledge on mechanisms underlying gestational diabetes that benefit public health.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by α-synuclein-rich neuronal inclusions. Recent genome-wide associated studies (GWAS) and epidemiological studies have identified multiple candidate genes and environmental factors, respectively, which can modify PD risk. Studying polygenic interactions with environmental factors has been difficult due to the lack of a model system. However, studies have hinted at a complex relationship between α-synuclein, the genetic risk factors, and environmental factors. In our preliminary data, we have established a multiplex model using the Drosophila model of PD. In this model, we express human α-synuclein, simultaneously modify GWAS candidate genes in neurons, and expose adult flies to rotenone. Using a combination of scalable techniques in this model, we identified novel interactions among α-synuclein, environmental factors, and GWAS genes. The overarching hypothesis is a multiplex model, in combination with iPSC-derived neurons, can be used to identify and study the mechanism of novel gene-environment interactions. Further, this model system will identify potential drug targets that can modify the gene-environment interactions. In Aim 1, a series of experiments, including super-resolution microscopy and iPSC-derived tyrosine hydroxylase (TH) neurons, will be performed to characterize the interaction among LRRK2, rotenone, and α-synuclein, which was identified using the multiplex model. These experiments will be performed in the laboratory of primary mentor Mel B. Feany. Aim 2 will involve understanding the mechanism of interactions among LRRK2, rotenone, and α-synuclein. Previous studies and preliminary experiments have shown that actin hyperstabilization plays a central role in regulating neurotoxicity. Herein biochemical, immunohistological, and neurotoxicity assays will be performed in Drosophila and iPSC-derived TH neurons (obtained disease-causing LRRK2-G2019S and protective LRRK2-R1398H iPSCs) to study the role of actin dynamics in regulating this gene-environment interaction. These experiments will be performed in Dr. Feany's lab. In the independent R00 section, a druggable target that can modify the interaction among LRRK2, rotenone, and α-synuclein will be identified. Further, we will screen for other PD-related neurotoxicants that interact with LRRK2 and α-synuclein through actin hyperstabilization. We will genetically and pharmacologically inhibit MRCKα, a kinase that can regulate actin hyperstabilization, in flies, iPSC-derived neurons, and a mouse model. My neurotoxicology and neurodegeneration training will be facilitated by didactic courses and participation in Clinical Pathological conferences at Harvard Medical School and the Exposome boot camp at Columbia University organized by co-mentor Gary Miller. This project may elucidate a novel model system that can be used to identify and study the mechanism of gene-environment interactions. The training that I undertake will enable me to transition to independence and lead a laboratory investigating the molecular mechanisms of gene-environment interactions in neurodegenerative disorders.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract Drinking problems and sleep problems each cause significant loss at individual and societal levels. Insomnia in particular is highly prevalent in patients with alcohol use disorder, is prospectively associated with the development of alcohol use disorder and contributes to poorer recovery prognosis following alcohol treatments. Insomnia, therefore, represents a modifiable risk factor for negative outcomes associated with alcohol use along the full continuum of alcohol use problems. Accordingly, the project proposes to improve sleep with an insomnia intervention tailored to individuals who meet widely accepted definitions for hazardous alcohol use as well as diagnostic criteria for insomnia disorder. This is a treatment development study that will adapt cognitive behavioral therapy for insomnia for adults engaged in hazardous use of alcohol. We propose an iterative approach to development, refinement, and preliminary examination of the utility of a telephone-delivered, 4-session version of cognitive behavioral therapy for insomnia adapted to hazardous alcohol users. The project will begin with a small, open label pilot to develop and refine procedures for administering the intervention. Then, we will conduct a small, randomized trial comparing the intervention to a sleep and alcohol education control condition. As this is a treatment development award, we will assess a number of intervention and study design feasibility domains in preparation for designing a larger study. We will also assess the effects of the intervention on alcohol use, sleep and mood by measuring these outcomes at baseline, post-treatment, and at 3- and 6-month follow up assessments. In sum, this proposal is the first step in a program of research that intends to use sleep as a lever to alter the course of hazardous alcohol use. Here, the first step is to adapt an already successful insomnia intervention to a unique population and conduct a preliminary test of that interventions’ acceptability to patients, fidelity by therapists, and effects on drinking, sleep and mood. If study results are promising, we will use these data and information gleaned about the study methods to pursue the next phase of research: designing and conducting a definitive study to test our insomnia intervention’s capacity to decrease alcohol use and modify the course of hazardous alcohol use.

NIH Research Projects · FY 2026 · 2023-07

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors that function as dimers. While mGluRs are known to form homodimers, more recent work has shown that they can also heterodimerize, but not promiscuously. Because mGluRs exhibit widespread expression in the brain and regulate excitability and plasticity, they have become candidates as druggable targets for a variety of pathologies. To date however, excitement generated by preclinical data has not resulted in mGluR-targeting therapies in the clinic, despite a wealth of available ligands with good selectivity targeting these receptors. Our recent work examining mGluR2/4 heterodimers provides a possible explanation: ligands that are highly efficacious when targeting homodimeric receptors are often without effect when the same receptor is expressed as a heterodimer with another mGluR. Further complicating matters, these changes in pharmacological responses observed in mGluR2/4 heterodimers are not generalizable to all mGluR heterodimers, or even all mGluR2 containing heterodimers. Thus, to understand how any mGluR ligand will function in the brain, we must examine the pharmacological responses of each possible heterodimer pair in isolation. But this is complicated because every mGluR can also form homodimers, so any pair of expressed mGluR will have an unknown propensity to homo- and heterodimerize. To solve this problem, we have designed a novel dimer composition control system using a combination of ER retention sequences paired with orthogonal, split inteins, self-excising protein sequences, that will allow expression of pure populations of nearly wild type mGluR dimers of known composition. We plan to generate a comprehensive ligand vs. mGluR dimer atlas to be used to not only aid in interpretation of experimental data but also to improve therapeutic strategies targeting mGluRs for a range of pathologies. To accomplish these goals, we will pursue the following Specific Aims: 1, T To build and characterize the full complement of tagged mGluRs using the split intein-ER retention strategy, 2, To employ an adapted CODA-RET approach to obtain parallel heterodimer specific G protein recruitment data, and 3, To systematically assess the pharmacological responses of each probable mGluR dimer pair to selective agonists, competitive antagonists, PAMs and NAMs.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT Myopia is a refractive error type of eye disorder where light is focused in front of the retina, requiring optical corrections to recover the resulting loss in visual acuity. It is a broadly significant health condition: it is estimated that by 2050 50% of the world population will be myopic. Moreover, even if myopia is compensated with spectacles, contact lenses or surgery, high myopia is linked to a higher risk of retinal detachment, glaucoma, and cataract. Strategies to halt the progression of myopia are therefore an urgent need. Myopia arises from a mismatch between ocular axial length and optical power, but the signals that prompt excessive eye growth are not well understood. Among various strategies developed for myopia control, the use of multifocal contact lenses (MCL) is gaining significant traction. A generalized working principle behind MCL design is that induced myopic defocus in the peripheral retina is protective for foveal axial growth. Initial clinical trials report encouraging reduction of myopia progression in children fitted with MCLs (generally, center-distance and high add powers) however they are still far from the desired effectiveness. Unlike MCL for presbyopia, MCLs for myopia control are prescribed on subjects that can accommodate. However, we currently do not have a good understanding of how accommodation interacts with MCLs in determining retinal image quality. This is a critical gap in knowledge as accommodation affects the very central feature of MCL design — the degree and the sign of retinal defocus. For example, depending on MCL design and individual physiological parameters, some subjects could rely on the near zones of the MCL for near vision, potentially exposing the retina to hyperopic defocus and triggering eye growth. We, a diverse team of optical engineers, physicists and neuroscientists will make use of adaptive optics simulation technologies and psychophysical paradigms to map non-invasively MCL lens patterns onto the subject’s pupil and systematically address key outstanding questions on the interplay between MCL design, accommodation, image quality and visual function with MCLs in young myopes. The group has previously developed adaptive optics technologies to test presbyopia corrections, novel methods based on wavefront sensing to quantify the accommodative response, IOL designs for presbyopia and psychophysical paradigms suited for young subjects. We are now using these capabilities, expanded to binocular simulation and testing, to understand factors underlying accommodative and binocular mechanisms of MCL-based interventions to slow down myopia. The long-term goal is to develop a mechanistic understanding that can help guide the design and personalization of MCL myopia control interventions. We plan to 1) determine the accommodative response with multifocal patterns in young myopes and emmetropes, 2) quantify the effects of various multifocal designs on foveal visual function and 3) test the role of accommodative response on the outcome success in children that have been clinically treated with multifocal contact lenses

NIH Research Projects · FY 2025 · 2023-07

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors that function as dimers. While mGluRs are known to form homodimers, more recent work has shown that they can also heterodimerize, but not promiscuously. Because mGluRs exhibit widespread expression in the brain and regulate excitability and plasticity, they have become candidates as druggable targets for a variety of pathologies. To date however, excitement generated by preclinical data has not resulted in mGluR-targeting therapies in the clinic, despite a wealth of available ligands with good selectivity targeting these receptors. Our recent work examining mGluR2/4 heterodimers provides a possible explanation: ligands that are highly efficacious when targeting homodimeric receptors are often without effect when the same receptor is expressed as a heterodimer with another mGluR. Further complicating matters, these changes in pharmacological responses observed in mGluR2/4 heterodimers are not generalizable to all mGluR heterodimers, or even all mGluR2 containing heterodimers. Thus, to understand how any mGluR ligand will function in the brain, we must examine the pharmacological responses of each possible heterodimer pair in isolation. But this is complicated because every mGluR can also form homodimers, so any pair of expressed mGluR will have an unknown propensity to homo- and heterodimerize. To solve this problem, we have designed a novel dimer composition control system using a combination of ER retention sequences paired with orthogonal, split inteins, self-excising protein sequences, that will allow expression of pure populations of nearly wild type mGluR dimers of known composition. We plan to generate a comprehensive ligand vs. mGluR dimer atlas to be used to not only aid in interpretation of experimental data but also to improve therapeutic strategies targeting mGluRs for a range of pathologies. To accomplish these goals, we will pursue the following Specific Aims: 1, T To build and characterize the full complement of tagged mGluRs using the split intein-ER retention strategy, 2, To employ an adapted CODA-RET approach to obtain parallel heterodimer specific G protein recruitment data, and 3, To systematically assess the pharmacological responses of each probable mGluR dimer pair to selective agonists, competitive antagonists, PAMs and NAMs.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT This proposal details a five-year research and career development plan with a scientific focus on autoimmunity-associated B cells (ABC), a subset of cells that is expanded during lupus disease activity and can differentiate into autoantibody-producing plasma cells (PC). These cells are found in the kidneys of lupus nephritis patients. The long-term objective of the study is to understand signals that regulate the development of ABC and autoreactive PC in lupus nephritis patients in hopes of identifying new therapeutic targets. Preliminary data suggest that ABC develop after B cell activation in the presence of interferon-gamma (IFN-γ) and interleukin 21. ABC are hyper-responsive to type III interferon (IFN-λ). PC differentiation can be promoted by IFN-λ in healthy B cells. In this application, Aim 1 will determine how interferon lambda (IFN-λ) promotes ABC differentiation to PC by examining epigenetic and gene expression changes in B cells treated with IFN-λ. Aim 2 will define the relationships between ABC and their cellular neighbors in the renal microenvironment. In particular, the developmental relationship between ABC and other B cells in the kidneys of lupus nephritis patients will be examined using transcriptomic approaches. A gene expression map of the cellular neighbors of ABC and PC in the lupus nephritis renal microenvironment will be created using spatial transcriptomic analysis. This will identify factors that may be promoting the development of autoimmunity in lupus nephritis. This project will allow Dr. Jennifer Barnas, MD, PhD to develop her skill set in molecular techniques such epigenomics, spatial and single cell transcriptomics and biostatistical anlaysis of these large data sets under the mentorship of Dr. Jennifer Anolik, an expert in lupus B cell biology, Dr. Martha Susiarjo, an expert in epigenetic regulation of disease, and Dr. Andrew McDavid, a computational biologist with extensive experience in single- cell analysis of immune cells. Dr. Barnas will use these techniques to develop an independent research program at University of Rochester and apply for NIH R01 funding to study B cell activation pathways and PC development in autoimmune disease. This work will inform the origin of ABCs and establish whether pathways revealed by in vitro studies are functioning in clinically relevant tissue samples.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT Bacterial keratitis (corneal infection) is a vision threatening disease and contributes significantly to world blindness. The important human pathogen, Staphylococcus aureus, is a predominant cause of keratitis, causing aggressive infections that often lead to irreparable ocular tissue damage. While much attention has been given to understanding the pathogenesis of S. aureus in non-ocular sites of infection such as sepsis, endocarditis or osteomyelitis, surprisingly, relatively little is yet known about the bacterial virulence factors that govern S. aureus keratitis. In the absence of this knowledge, it is impossible to understand the fundamental pathogenesis of S. aureus keratitis or rationally design therapeutics to treat this blinding disease. Accordingly, the central goal of this proposal is to identify and characterize S. aureus genetic determinants that modulate keratitis. Given the unique environment of the ocular surface and cornea with respect to tear film composition, sheer forces generated from blinking, and relative immune privilege, we hypothesize that there will be a unique set of virulence factors that drive keratitis. Our laboratory has developed innovative tools that will allow, for the first time, the ability to prospectively identify and validate the comprehensive set of S. aureus virulence factors that play a role in BK pathogenesis. Our discovery pipeline leverages state of the art genomics and bioinformatics including whole genome sequencing, Tn-seq and RNA-seq to generate high priority leads for mechanism of action studies, as well as the evaluation of biologic significance in our murine model of keratitis. We have successfully used this approach to identify and validate enterotoxins, secreted bacterial proteins well known for their cytotoxicity and ability to modulate the host immune response, as important in mediating keratitis. Thus, this proposal will build on this exciting preliminary data to develop an expanded list of the key drivers of keratitis. Ultimately this work will directly advance our understanding of S. aureus keratitis pathogenesis and as such, may be foundational in supporting strategies for the therapeutic intervention of this debilitating disease.

NIH Research Projects · FY 2024 · 2023-06

Project summary/abstract Eye movement abnormalities in schizophrenia (SZ) have been documented for over 100 years, yet it remains unknown whether fine-scale eye movements differ in this population. This is unfortunate because the neural structures underlying oculomotor control are well-documented, implying that group differences could shed light on the underlying illness pathophysiology. Moreover, establishing fine-scale eye movement differences could in principle provide a distinguishing biobehavioral marker, which in turn could be used for differential diagnosis or clinical prediction. Additionally, fine-scale eye movements contribute to everyday activities, such as recognizing facial expressions at a distance, reading, and discriminating fine spatial stimuli. Therefore, it is conceivable that abnormal micro- eye movements could be associated with—and even causally related to—impairments of these visual functions in psychosis. Thus, a major goal of the research described in this proposal is to establish that fine-scale eye movements do indeed differ among those with psychosis (Aim 1). These differences will be assessed in people with a psychotic disorder and in well-matched healthy adults during steady fixation and in the presence of complex foveal stimuli. In contrast to possibly all prior psychosis studies, we will use a high- precision digital Dual Purkinje Image Eye-Tracker (1 arcmin resolution) with a head-stabilizing helmet to minimize small head motions, and frequent re-calibrations throughout data collection. Additionally, a custom-made system for gaze contingent display control, will enable the implementation of a state-of-the-art calibration procedure, which effectively reduces the gaze localization error to less than 5 arcmin. Since visual acuity and reading are often impaired among psychosis patients and may even portend a future psychotic disorder, we will attempt to generate group differences with tasks that probe these same processes. Moreover, to establish a reference baseline for fine oculomotor behavior and to consider hitherto unnoticed problems in fixational stability, we will assess small eye movement differences during steady fixation without an active task. Aside from assessing whether micro- eye movement differences characterize psychosis, we will also assess their relationship to visual perceptual deficits (Aim 2). In particular, we will probe for correlations between microsaccade characteristics and the magnitude of visual acuity and reading deficits. We will also consider whether trials with a higher rate of microsaccades are related to impaired reading or impaired visual acuity compared to trials without such eye movements, and whether image stabilization on the retina (which removes the benefits of microsaccades) worsens acuity more for controls than for patients. Findings will inform our understanding of how eye movement differences in psychosis are related to perception at the micro-scale, which in turn can suggest novel treatments or interventions for improving poor visual acuity and reading.