University Of Rochester

NIH Research Projects · FY 2026 · 2024-01

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease that affects approximately 1.5 million people in the U.S. Evidence exists for a genetic component to ME/CFS based on familiality studies performed by us and others, as well as twin studies. While multiple candidate ME/CFS genes have been identified by candidate gene and genome-wide association studies, no validated functional ME/CFS variants have been identified. As there is strong evidence that ME/CFS is heritable, we plan to overcome the limitations of prior genetic studies by studying related ME/CFS cases in high-risk ME/CFS pedigrees, or individuals with likely a stronger genetic contribution to ME/CFS. We propose to identify rare, candidate ME/CFS variants using a family-based, discovery design defined by affected cousins. We will leverage high-risk ME/CFS pedigrees with a statistical excess of ME/CFS that we have identified in the Utah Population Database, a database that links genealogy data back to the 1800s to medical record data, to identify high-risk ME/CFS pedigrees that include an affected cousin pair. We will recruit 50 unique, affected cousin pairs (100 total subjects) as well as other affected family members. For the variant discovery phase, we will perform whole genome sequencing of the affected cousin pairs to identify shared, rare variants. We will then evaluate the identified genes and regulatory variants in other existing ME/CFS genetic data sets, including DecodeME and All of Us. We will test for segregation of biologically plausible variants in other affected relatives, examine the gene’s role in pathways with other ME/CFS candidate genes, perform in silico protein prediction of the identified candidates, and characterize the studied ME/CFS cases and their pedigrees for risk of ME/CFS associated comorbid conditions (e.g., fibromyalgia). Our expected outcome is the identification of a set of ME/CFS predisposition variants/genes with segregation evidence in the families in which they were observed and validation in other datasets. Study of familial ME/CFS cases complements prior genome-wide association studies (GWAS) through identification of rare variants that contribute to ME/CFS. Our work has the potential to enhance understanding of the pathophysiology of ME/CFS, and it has the strong likelihood of providing targets for ME/CFS treatment and earlier diagnosis of ME/CFS.

NIH Research Projects · FY 2026 · 2024-01

Acute kidney injury (AKI) is a frequent complication among critically ill children and is independently associated with increased morbidity and mortality. While AKI identification remains largely reactive, early prediction of AKI provides opportunities for mitigation strategies to improve patient outcomes. Existing pediatric AKI risk prediction models are primarily driven by single-center data with limited translation into clinical decision support (CDS) at the point of care. While CDS systems are designed to improve care quality and patient safety, they have failed to make meaningful improvements while contributing substantially to provider alert fatigue. This is especially true in pediatric critical care, where alerts and alarms are ubiquitous and often ignored. Disregarding important alerts may lead to patient harm. The addition of machine learning methods to augment decision support has been the focus of substantial hype, however clinicians remain skeptical over concerns such as utility, credibility, and validity. Within this clinical context, this application addresses two specific knowledge gaps. First, without validated and generalizable prediction tools, the best method for identifying patients at risk for developing AKI in the pediatric ICU (PICU) is unknown. Second, current CDS systems are burdensome and there exist no clear recommendations on useful, usable implementations for AKI decision support. The long- term goal of my research program is to improve the care and outcomes of critically ill children with acute kidney injury while reducing provider alert frustration through personalized, shareable CDS. As a step toward this goal, this career development award will demonstrate success of an integrated CDS pipeline for external validation, cutting-edge technology to facilitate distributed implementation, and usability assessment. The research aims of this proposal are to: 1) establish baseline external validity of an existing AKI prediction model and extend with new predictors to improve accuracy within two novel multi-center datasets, each with >80,000 PICU encounters; 2) overcome interoperability barriers using Fast Healthcare Interoperability Resource (FHIR)-based EHR technology and prospectively evaluate this implementation’s performance; and 3) implement principles of user-centered design to improve clinician acceptance. With the support of my mentoring committee, I will gain advanced knowledge and develop expertise in user-centered design, qualitative methodology, clinical trial design, and implementation science in decision support. This work is innovative as it leverages two brand-new, highly granular datasets to achieve external validation, implements an AKI prediction tool using new FHIR-based technology, and utilizes mixed-methods cognitive informatics tools to perform usability testing. However, the truly innovative aspect of this work is the integration of all three aims into a single pipeline. This work provides the foundation for a prospective clinical trial evaluating clinical effectiveness, as well as incorporation of biomarkers and physiologic data streams, and the development of evaluation measures for decision support systems.

NIH Research Projects · FY 2026 · 2023-12

The objective of this application is to develop advanced multiview (MV) deconvolution tomography of cleared tissue using high resolution two photon microscopy (TPM) and utilize it to shed new mechanistic insights about the myofibroblast microenvironment in fibrovascular peritendinous adhesions. Advances to our understanding of the cellular and molecular events involved in tendon adhesion using transgenic mouse models, which could lead to disease modifying therapies, are often hindered by reliance on 2D histology sections and single-cell analysis techniques, neither of which can put cellular interactions or molecular processes into their 3D spatial context. Alternatively, microscopy techniques such as TPM and light sheet microscopy (LSM) can provide spatial context, but struggle to simultaneously provide the throughput, contrast and resolution required to map the cellular processes involved in tissue healing over physiologically relevant spatial and resolution scales and so are not widely used in tendon biology. To overcome these limitations, we have developed protocols for immunolabeling and tissue clearing of tendon injury models and demonstrated molecular and second harmonic generation (SHG) imaging of tendon in situ. Our proposed advances in detection electronics and imaging enable two photon microscopy two orders of magnitude faster than conventional microscopes and multicolor imaging at rates comparable to or exceeding LSM. Finally, we propose a novel approach to MV deconvolution, which simultaneously acquires multiple angular views to decrease focal volumes by an order of magnitude, enabling isotropic resolution tomographic imaging of tissues. We combine these technical advances into a high throughput platform for 3D spatial immunophenotyping and proteomics and then apply it to understanding the complex mosaic of vascular and inflammatory cells, extracellular matrix (ECM), and signaling (TGF- β1/mTOR) in the myofibroblast microenvironment, to shed new mechanistic insights that could lead to discovery of cellular and molecular targets for biologic therapies. The aims for this proposal are: Aim 1 will develop a new approach to tomography using MVD based on simultaneously imaging two angular views that will accelerate imaging and reduce the extreme computational complexity required to fuse two sequentially acquired volumes together. Eight channel acquisition will enable dense spectral multiplexing of probes and a deconvolution, enabling efficient quantification of spatial relationships between reporters at sub-wavelength scale. Aim 2 will apply MV-TPM to tomographically profile the myofibroblast microenvironment to build spatial cellular interaction networks of innate and adaptive immune cells within the vascular inflammatory microenvironment of myofibroblasts. Aim 3 will utilize MV-TPM imaging to map TGF-β1/mTOR signaling in the myofibroblast microenvironment and subsequently inhibit it to validate its role in adhesions. When completed, the proposed work will develop a high throughput 3D spatial immunophenotyping and proteomics imaging platform to shed novel mechanistic insights and discover new biologic therapies.

NIH Research Projects · FY 2026 · 2023-12

Although new treatments have improved outcomes in rheumatoid arthritis (RA), disability remains high, most patients have ongoing disease activity, and lasting remissions are rare. A major gap in the field is the elusive identity of pathogenic cells driving persistent inflammation and bone erosion. Our lab focuses on the identification of pathogenic B cell subsets in RA, which might drive persistent and aggressive disease. We reported that B cells promote bone erosion by RANKL/TNF-mediated differentiation of monocytes to osteoclasts (OCs) and TNF/CCL3- mediated inhibition of osteoblasts (OBs). Importantly, synovial B cells express much higher levels of these bone pathogenic factors. More recently, we identified a subset of B cells expressing the classic T cell lineage defining transcription factor T-bet enriched in the RA synovium and correlating with disease severity and the abundance of SLAMF7 pro-inflammatory monocytes. We find that synovial T-bet B cells express cytokines influencing OCs/OBs, SLAMF7, and the T- bet inducible chemokine receptor CXCR3. Remarkably, mouse B cells lacking T-bet did not activate OCs or inhibit OBs. Based on our results, the central goal of this proposal is to define how T-bet influences synovial B cell states, mediates B cell-driven bone effects on OCs/OBs, and coordinates B cell pathogenic functions in RA target tissue, contributing to joint erosion and synovial inflammation. We hypothesize that T-bet promotes pathogenic B cell functions in the RA synovium via acquisition of a pro-inflammatory cytokine/chemokine program that orchestrates CXCR3-dependent B cell migration to the synovium and enhances SLAMF7-dependent monocyte activation and bone erosion. We will use deeply characterized RA patient cohorts, high- resolution single cell transcriptomic and spatial analysis of joint target tissue, and novel animal models to track T bet B cells and selectively and conditionally delete T-bet in B cells of mice with collagen induced arthritis. The combination of preclinical and translational studies will facilitate the mechanistic interrogation of T-bet B cell functions in RA via 3 SAs: 1) Determine the mechanisms by which B cell T-bet promotes RA joint erosion; 2) Assess how T-bet B cells impact RA synovial monocyte activation; 3) Examine the role of T-bet in B cells during inflammatory- erosive arthritis in mice. By detailing the functional impact of T-bet B cells, innovative treatments targeting their activity may be a novel path towards preventing bone erosion and conferring lasting clinical remissions.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY / ABSTRACT People born preterm are at risk for developing pulmonary hypertension (PH) and heart failure that will profoundly shorten their lifespan. This is an emerging epidemic of growing concern because conservative estimates suggest there are 4 million survivors of preterm birth living in the United States, 40% of which have subclinical PH. The risk of developing disease is highest in adults born extremely preterm and thus directly associated with their need for supplemental oxygen (hyperoxia) at birth. But how hyperoxia at birth triggers disease, and whether PH and heart failure are integrated or separate diseases is not known. We propose to fill this gap in knowledge using lungs of preterm infants, adults who were born preterm, and an established mouse model wherein hyperoxia between postnatal days (PND) 0-4 causes PH via three distinct stages. The first or priming stage occurs when the lung is exposed to hyperoxia. During this time, hyperoxia stimulates endothelial expression of angiotensin converting enzyme (ACE) responsible for producing the vasoconstrictive peptide angiotensin (Ang) II and driving PH when uncontrolled. Hyperoxia also suppresses proliferation of cardiomyocytes lining the pulmonary vein and extending into the left atrium, resulting in left atrial dilation and insufficient filling of the left ventricle. This is followed by a latency period where ACE and Ang II levels continue to increase even though the lung is no longer in hyperoxia and the loss of atrial cardiomyocytes causes progressive diastolic dysfunction. The third or disease stage is characterized by high NADPH oxidase (NOX) 3 in pericytes, vascular remodeling and congestion, and mortality. The high expression of NOX3 in pericytes was unexpected and may explain why hyperoxia causes post-capillary remodeling, thus distinguishing it from pulmonary arterial hypertension (PAH) where smooth muscle cells express NOX4. Building off these findings, we hypothesize neonatal hyperoxia stimulates expression of ACE in pulmonary endothelial cells, which persists and increases because of ongoing endothelial injury and diastolic dysfunction, and causes PH when it dysregulates Ang II and NOX3 later in life. We will determine how hyperoxia regulates ACE in pulmonary endothelial cells (Aim 1), how NOX3 contributes to vascular remodeling (Aim 2), and whether suppressing ACE/Ang II signaling effectively reduces PH and diastolic dysfunction caused by hyperoxia (Aim 3). Understanding how hyperoxia causes cardiovascular disease could advance new concepts on how to best manage it in people born preterm.

NIH Research Projects · FY 2025 · 2023-12

ABSTRACT The exon junction complex (EJC) is deposited by the spliceosome upstream of exon–exon junctions during the process of pre-mRNA splicing. If an exon–exon is sufficiently downstream of either a normal termination codon or a premature termination codon (the latter of which can be generated by splicing errors or frameshift or nonsense mutations), then EJC-dependent nonsense-mediated mRNA decay (NMD) is triggered. NMD destroys aberrant mRNAs and regulates the normal gene expression of ~5-10% of unmutated mRNAs. I recently discovered that AKT, a central cell signaling kinase, is a constituent of an alternative EJC containing CASC3 (the AKT EJC) but not UPF2 or RNPS1 (in the canonical UPF2 EJC). AKT functionally replaces UPF2 in UPF2- independent NMD by activating the key NMD factor UPF1. AKT does so by phosphorylating UPF1; UPF2 does so by binding UPF1. I found that insulin-mediated activation of AKT signaling increases AKT EJCs at the expense of UPF2 EJCs, which is the first example of cell-signaling mediated regulation of EJC composition. These findings are important because AKT signaling and NMD, which converge at the EJC, are both dysregulated in some cancers, Fragile X syndrome, and type 1 and 2 diabetes. Despite its importance, there are many remaining uncertainties about how EJCs are formed, including: when and how the commitment to either the AKT EJC or UPF2 EJC is made; the mechanism by which insulin increases AKT EJC formation; if insulin increases and/or alters EJC binding at exon−exon junctions; if spliceosomes are involved in alternative EJC formation; the choreography of steps involved in core EJC formation, presumably on the spliceosome; and how EJCs are transferred to upstream of exon−exon junctions on newly spliced mRNA, to name a few. In this proposal, I will start addressing some of these uncertainties while receiving training in concepts and techniques from my mentoring team so as to continue pursuing these questions in my own lab. In Aim 1 (K99), I will test (i) the precursor-product relationship between UPF2 EJCs and AKT EJCs (while learning how to generate and use HEK293T cells engineered for auxin-induced degradation of EJC proteins), (ii) if the insulin-promoted increase in AKT EJCs and decrease in UPF2 EJCs requires splicing, and (iii) if insulin signaling increases and/or alters the number of EJC-bound exon–exon junctions (while learning RIP-Seq). In Aim 2 (K99), I will identify and define any insulin-promoted post-translational modifications (PTMs) on EJC proteins that promote the increase in AKT EJCs and decrease in UPF2 EJCs (while learning mass spectrometry (MS)-based proteomics and PTM profiling). In Aim 3 (R00), I will apply my background in mechanistic biochemistry and training in new techniques to build an independent research program aimed at elucidating yet-undetermined molecular mechanisms in EJC biology, starting by defining which splicing factors are needed for core EJC assembly, which splicing factors, if any, are involved in alternative EJC formation, and for the latter, whether PTMs of splicing factors are involved.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Black men who have sex with men (BMSM) continue to experience disproportionate burdens of HIV compared to other MSM subgroups in the US. This disparity is exacerbated by the multilevel intersectional stigmas that BMSM face, including those based on race, same-sex attraction/behavior, and presumed HIV-positive status, which results in high levels of psychological distress and poor HIV prevention outcomes. Resilience is a critical component in studies to mitigate the negative impact of multilevel intersectional stigmas on HIV prevention uptake among BMSM. However, existing resilience measures are designed for non-marginalized populations and are inadequate for assessing BMSM's resilience to multilevel intersectional stigmas. To address this gap, we propose to develop a novel, BMSM-specific, multidimensional stigma-resilience scale that captures the nuanced resiliency mechanisms countering intersectional stigmas across multiple socioecological levels. The proposed study will be conducted in Nashville, an HIV hotspot in the Southern US, where growing HIV burdens and intersectional stigmas among BMSM have been identified. The study has two specific aims. In Aim 1, we will define the mechanisms of multidimensional resilience that BMSM use to counter intersectional stigmas. We will conduct in-depth interviews with 30 HIV-negative BMSM in Nashville, guided by a conceptual framework that integrates the Socioecological Model, Resilience Theory, and Intersectionality Framework. We will explore how resilience is developed, manifested, and implemented to overcome intersecting forms (e.g., internalized, enacted, experienced, and anticipated) and types (e.g., stigma against racism, homosexuality, and presumed HIV status) of stigmas across multiple socioecological levels (e.g., individual, interpersonal, community and healthcare). Aim 2 will necessitate an iterative and systematic BMSM community engagement process to develop and validate a new multidimensional stigma-resilience measure tailored for BMSM. Insights from Aim 1 will help inform the adaptation of an existing scale (e.g., Multilevel Resilience Measure for Black Adults Living with HIV) to be contextually appropriate and stigma-focused for BMSM. We will then assess face validity and cultural relevancy through cognitive interviews with 10 BMSM, followed by conducting psychometric analyses with 250 BMSM to test the new scale's reliability (e.g., internal consistency) and validity (e.g., convergent, divergent, and predictive validity). The proposed study is significant in providing a robust, culturally-tailored stigma-resilience measure, which will inform our future R34/R01 study to develop and evaluate a multilevel resilience-based intervention to mitigate intersectional stigmas and HIV prevention uptake among BMSM. Additionally, the new BMSM Multidimensional Stigma-Resilience Scale will provide the groundwork for future studies to refine and adapt the scale for resilience-focused research conducted with other marginalized populations (e.g., Latino/Hispanic MSM; transwomen of color).

NIH Research Projects · FY 2025 · 2023-09

Under almost all conditions using any method, the levels of gene transfer to any cell or tissue are low because many barriers exist for the efficient delivery of genes to cells. The primary goal of our laboratory is to identify and overcome the intracellular barriers to promote effective gene delivery and therapy. Exogenous viral or non- viral DNA must cross the plasma membrane, travel through the cytoplasm and the cytoskeletal networks, cross the nuclear envelope, localize to specific regions within the nucleus, and be transcribed in order for gene therapy to be successful. We have shown that once in the cytoplasm, plasmids carrying DNA nuclear targeting sequences (DTS) that are required for nuclear import in non-dividing cells rapidly associate with transcription factors that mediate movement along microtubules and across the nuclear envelope. NF-kB is one such factor that binds to several ubiquitously active DTSs and is required for DNA nuclear import, but in the cytoplasm it is maintained in a sequestered state, unable to bind DNA. The question then is how is NF-kB activated to bind to plasmids and mediate their cytoskeletal movement and nuclear import? In the case of NF-kB, a major pathway for its activation is through a set of cytoplasmic dsDNA sensors, such as cGAS-STING, that are part of the innate immune system and drive inflammatory responses. When dsDNA binds to cGAS, signaling cascades are initiated that result in activation of key pro-inflammatory transcription factors (including NF-kB) and ultimately production of pro-inflammatory cytokines. Thus, a major focus in the gene therapy space has been to block activation of these sensors to reduce inflammation. However, we have observed that when cGAS is silenced, cytoplasmically injected plasmids fail to traffic to the nucleus. We hypothesize that limited activation of one or more of these sensors is actually needed for low level activation of key transcription factors in order to facilitate DNA nuclear import in non-dividing cells. If we can find ways to limit sensor activation, but not abolish it, this will allow for enhanced gene delivery with limited accompanying inflammation. We have also spent considerable effort detailing the distribution of plasmids inside the nucleus and have found that the subnuclear mislocalization of plasmids can affect their transcriptional activity. We have found that plasmids localize to discrete transcriptional domains within the nucleus based on the type of promoter (Pol I, Pol II, or Pol III) they carry and that when two different promoter types are placed on one plasmid, not only is the intranuclear distribution of the DNA different that either promoter type alone, but transgene expression is significantly reduced. We will dissect the pathways used for DNA movement within the nucleus and exploit them to improve transgene expression based on the subnuclear localization of the transfected DNA. Our specific aims are to (1) determine whether cytosolic dsDNA sensors are required for DNA nuclear import; (2) evaluate whether residence time of DNA in the cytoplasm affects sensor activation and transfection efficiency; and (3) characterize how subnuclear organization affects exogenous DNA expression.

NIH Research Projects · FY 2025 · 2023-09

The Focus: Localized provoked vulvodynia (LPV) is the most common cause of chronic dyspareunia (painful intercourse) in premenopausal women, and it remains a poorly understood disease. Existing therapies do not target the underlying causes, treatment is trial and error, and intervention typically escalates to cutting away the painful tissue surrounding the vaginal opening, the vestibule. This proposal aims to identify new targets to treat LPV pain in alignment with the missions of at least two NIH institutes by aiming to 1) improve women’s health care (NICHD) and 2) identify non-addictive targets for pain therapy (NIDA). The Premise: We discovered a non-classical inflammatory response in the vestibule of LPV patients that is a key contributor to LPV pain. The vestibule of LPV patients is hypersensitive to inflammatory stimuli, causing a response when one would not otherwise occur, which is characterized by high levels of proinflammatory mediators. There is a direct association between proinflammatory mediator levels and pain; fibroblasts taken from sites of exquisite pain produce the highest levels of these mediators, indicating the vestibule could be uniquely vulnerable and could be specifically targeted to resolve pain. We subsequently identified several candidates for therapeutic intervention (e.g. Dectin-1, Nuclear kappa factor B). However, targeting these would not completely alleviate proinflammatory signaling or might compromise host defenses. Our goal is to identify and validate new therapeutic targets for LPV pain therapy. Our supporting data strongly suggest that transient receptor potential vanilloid receptor 4 (TRPV4) and members of its signaling pathway represent promising and innovative therapeutic targets. We will confirm TRPV4’s role in LPV, validate the likely therapeutic targets, and in the process, enhance our mechanistic understanding of vulvodynia. Organizing Hypothesis: We hypothesize that targeting the TRPV4 pathway will reduce pro-nociceptive signaling in human fibroblasts and tissue and impart analgesia in mice Specific Aim 1: Elucidate the role of site-specific TRPV4 signaling differences to identify new therapeutic targets for LPV. Specific Aim 2: Explore the relationship between inflammation, TRPV4, and alterations in lipid profiles in LPV patients. Specific Aim 3: Validate TRPV4 and other identified targets using 3D tissue culture and an in vivo LPV model. Impact on the field: We plan to accomplish three goals: 1) identify and validate new targets for desperately needed non-invasive and efficacious vulvodynia therapies, 2) improve understanding of the vulvodynia mechanism, and 3) identify mechanisms likely conserved in other pain conditions by focusing on a ubiquitous signaling pathway (TRPV4) suspected to play a role in pain syndromes, targeting of which would be unlikely to result in adverse sequelae, including drug dependence disorders.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract People with fetal alcohol spectrum disorders (FASD) experience barriers to care and a lower quality of life (QOL). FASD- informed services and supports are lacking, especially during the transition to adulthood – a critical developmental period for QOL. The proposed project will develop and initiate testing of a scalable person-centered planning (PCP) intervention to support QOL for adolescents and adults with FASD. PCP is an evidence-based intervention for people with disabilities. Research suggests PCP is a good fit FASD; however, it has yet to be adapted or tested with this population. In this project, we will use rigorous implementation science frameworks to systematically adapt and test a PCP intervention for adolescents and adults with FASD. Given the importance of social support networks in PCP, we will also draw upon the growing literature and team expertise on social network interventions to inform the development and evaluation of the intervention. Consistent with other interventions in our lab, we expect the proposed intervention to leverage technology to offer scalable solutions to overcome current barriers to care. During the R61 phase of the project, we will use the implementation science framework “Intervention Mapping – Adapt” to carefully adapt the core principles and methods of PCP and social network interventions for FASD. This 6-step process involves a planning team of people with FASD and other stakeholders and careful attention to theory and logic models to guide adaptation decisions. Based on our preliminary logic models, self-determination theory is a strong fit with the proposed intervention that we are currently calling “Thrive.” Additional research activities in Aim 1 will also inform the planning group’s decision making as they refine logic models, determine intervention structure and components, develop materials, and finalize evaluation procedures. Specifically in Aim 1a, we will conduct individual and group interviews with adolescents and adults with FASD and members of their social networks to characterize key features of their support networks that may be amenable to intervention. Following initial production of intervention materials, we will complete two rounds of usability testing (5 participants per round) in Aim 1b to aid in refining intervention materials and evaluation procedures for the pilot randomized controlled trial (RCT) in the R33 phase. Following successful attainment of R61 milestones, we will conduct a type 1 hybrid effectiveness-implementation pilot RCT of the developed Thrive intervention (R33). This trial design involves gathering data on both effectiveness and implementation outcomes, which will help us optimize the intervention for future larger-scale trials and more rapid translation into community settings. We will recruit 60 adolescents and adults (ages 15-30) with FASD to participate in the pilot RCT, with random assignment to the 1) Thrive PCP intervention or 2) comparison group receiving social network and strengths assessments only. Although significance testing and effect sizes will be calculated, a larger emphasis will be placed on feasibility to guide the design of a fully powered larger-scale RCT R01 application. For example, in addition to the feasibility of the intervention itself, we will assess feasibility of recruitment and trial procedures, sensitivity of measures to intervention change, and intervention process. Our proposed PCP intervention has high potential to improve QOL of people with FASD at the critical transition to adulthood.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Young gay, bisexual, and other men who have sex with men (YGBMSM) make up 63% of the HIV prevalence (18%) among GBMSM in Ghana. Our previous studies show that intersectional stigma and discrimination (ISD) (around HIV, sex, and gender expression) and insufficient HIV knowledge impede HIV testing, PrEP, and ART (HPART) adherence among GBMSM. Those in slums face additional stigma associated with their communities; hence the social and economic barriers in slum communities can intertwine with ISD to exacerbate the risk of HIV infections and discourage HPART adherence among YGBMSM as they internalized stigma and avoid HCF. A status-neutral approach to HIV care can reduce ISD among YGBMSM and HCFs and improve HPART among YGBMSM. Yet, current HIV interventions and programs in Ghana have focused on separate services and predominantly address only testing. To this effect, we propose to adapt a multilevel intervention (LAFIYA, meaning wellness) to address ISD and HPART using status neutrality among YGBMSM and HCF in Ghanaian slums. LAFIYA showed early efficacy in increasing HIV testing/self-testing among HIV-negative or unknown- status YGBMSM in Ghanaian slums. Adaptation to a status-neutral intervention will extend its utility for HIV prevention and care by utilizing scientific advancements around intersectional stigma, HIVST, PrEP, and linkage to care. LAFIYA also mainly focused on GBMSM and involved providers from two facilities who received ISD reduction training. The proposed LAFIYA will include HCF-wide training, PrEP, and ART components. Guided by the eight steps of the ADAPTT-IT model, we will implement the two aims. 1) Adapt a multilevel intervention to address ISD and HPART using status neutrality among YGBMSM. We will conduct FGDs and in-depth interviews (IDI) with YGBMSM (n=70), GBMSM providers (n=20), and NURSES/HCWs (n=80) to deepen understanding of ISD, barriers, and facilitators of HPART adherence among YGBMSM. In a summative analysis, we will integrate realities from providers and YGBMSM to optimize the intervention potential to address ISD and increase HPART. 2) Test the preliminary efficacy of the intervention to address ISD and increase HPART adherence using CRT Design. We will assign YGBMSM (n=240) friend groups to control or intervention groups (n=120) and HCF(n=6) to control or intervention (n=3). The intervention arm will receive LAFIYA. We will collect 3, 6, and 9 months post-intervention data among YGBMSM (n=240) and HCWs(n=300). to measure HPART adherence (primary outcomes), ISD reduction, HIV and status-neutral knowledge (secondary outcomes), and acceptability, appropriateness, and feasibility of the intervention (implementation outcomes). We will conduct additional focus groups and interviews post-intervention among implementers and participants (sample sizes same as in AIM1) to evaluate their experiences with the intervention. The findings will inform intersectional stigma reduction and HIV status-neutral implementation strategies among YGBMSM, slums, SSA, and other high-risk populations in different settings.

NIH Research Projects · FY 2025 · 2023-09

Abstract Cardiovascular disease is the leading cause of death worldwide. Epidemiological studies indicate a biphasic association between alcohol and cardiovascular disease with frequent low-to-moderate consumption being protective, whereas heavy bingeing and chronic abuse is harmful. Currently lacking, however, is in-depth mechanistic knowledge of how different levels of alcohol impact arterial cells to ultimately dictate vessel pathology and disease progression. Most of the problems associated with cardiovascular disease begin with arteriosclerosis, a thickening and stiffening of the artery wall, that may progress to plaque development and blood flow blockage resulting in heart attack or stroke. Arteriosclerosis involves medial and intimal hyperplasia (i.e., intima-media thickening or IMT) due to the accumulation of ‘vascular smooth muscle-like’ a-SMA+ cells. Emerging evidence suggests that in response to injurious stimuli such as inflammation or disturbed flow, endothelial cells can transform into different cell types, especially myofibroblasts in a process known as endothelial-to-mesenchymal transition (EndMT), and thus contribute to intima-media thickening. Crucially, no information exists as to whether alcohol consumption, a common modifiable behavior and a known modulator of cardiovascular disease, might regulate endothelial transformation in this context, a question of considerable interest and the focus of our proposal. Based on our exciting preliminary data in human arterial cells and in a mouse model, we will use gain-and-loss of function approaches in vitro and in vivo, in combination with multi- color ‘Confetti’ reporter lineage tracing and single-cell RNA-sequencing analyses to test our hypothesis that alcohol has a biphasic effect on atherogenic stimuli-induced endothelial phenotypic transformation to differentially affect vessel homeostasis and arteriosclerosis and to elucidate the involvement of Notch in mediating these responses. Data generated will markedly increase our basic science understanding of how drinking affects cardiovascular disease, information that could be leveraged to inform novel treatments for this leading cause of morbidity and mortality.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The Integrator Complex (INT) is a 17-subunit machinery that associates with RNA polymerase II (RNAPII) and functions as a critical transcription regulator. It is essential for the 3'-end formation of a variety of non-coding RNAs and is a broad negative regulator of promoter-proximally paused RNAPII. Integrator subunit 11 (INTS11) houses the RNA endonuclease domain vital for Integrator to cleave nascent transcripts at all RNAPII loci, which is an important activity for transcriptional repression. Consistent with a fundamental role in Integrator function, INTS11 genetic variants are found to disrupt human development and give rise to a complex neurological syndrome. Similarly, genetic disruptions in two non-Integrator proteins that associate with INTS11 also impede neurodevelopment, including BRAT1, where variants cause a lethal neonatal multi-focus seizure syndrome and cerebellar ataxia, and WDR73, where variants cause Galloway-Mowat syndrome that is characterized by psychomotor impairment, hypotonia, and seizures. WDR73 and BRAT1 associate with INTS11 and INTS9 but are primarily cytoplasmic proteins distinct from the complete 17-subunit nuclear Integrator Complex. Notably, nothing is known about how these proteins bind INTS11 or their role in nuclear Integrator function – especially in the context of neuronal cell fate and fitness. Using biochemical and structural approaches, we generated cryo-EM structures of the INTS11-BRAT1 binary complex and the INTS9-INTS11-BRAT1 ternary complex. Surprisingly, we also find that optimal INTS11 function within the nuclear Integrator Complex requires interaction with these cytoplasmic proteins as cells lacking BRAT1 or WDR73 accumulate uncleaved Integrator substrates. Altogether, these preliminary studies generate a provocative model whereby cytoplasmic BRAT1 and WDR73 are required to ‘license’ INTS11 for its nuclear activity, which is critical to maintaining proper neuronal function in humans. To test this hypothesis, we propose these Specific Aims: Specific Aim 1. Structurally and biochemically characterize the INTS9, INTS11, BRAT1, and WDR73 complexes. Specific Aim 2. Probe the function of BRAT1 and WDR73 in Integrator-mediated gene regulation. Specific Aim 3. Decipher the role of the INTS11-containing complexes in neural cells.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Child maltreatment (CM) is a broad-ranging risk factor associated with compromised development and maladaptation. Yet, there is vast heterogeneity in the experience of CM and its developmental outcomes. Several of the field’s most pressing developmental questions involve exploring such heterogeneity. However, investigating risk heterogeneity in CM populations requires sensitive longitudinal studies of high-risk, hard-to- reach subjects with adequate power to detect unique subgroups who differ in the experience and consequences of CM—such studies are costly, arduous, and rare. This project aims to address this gap. The overall objective of this project is to apply Integrative Data Analysis (IDA)—a principled set of methodologies and statistical techniques used to conduct simultaneous analysis of raw data pooled from multiple datasets—as a method to address questions about risk heterogeneity that may not be addressed through individual CM studies alone. This project will use IDA to pool data from 7 NIH-funded CM cohorts that used gold-standard methods to examine the development of long-term CM sequelae across biopsychosocial domains. Pooling original data from multiple CM studies stretches the developmental period under observation, generates a more heterogenous sample, and increases statistical power to examine important sources of risk heterogeneity. IDA will yield an integrated sample (N = 2,898) that includes assessment of an array of biopsychosocial processes from ages 4 through 40. The IDA dataset will be used to address three aims: A1) determine how heterogeneity in CM exposure (i.e., variation in types, developmental timing, and chronicity of exposure) differentially influences developmental sequelae; A2) identify heterogeneity in the developmental outcome trajectories of CM survivors and examine which features of CM exposure are associated with specific trajectories; A3) explore how CM exposure and subsequent developmental processes differ based on racial/ethnic heterogeneity. This project is innovative because it will leverage $25 million of NIH investment in CM research to unlock the constraints of isolated studies, creating a pooled source of CM data that is more powerful and diverse than any individual cohort, maximizing the value of complementary efforts in the field. This contribution will be significant because it will help to parse risk heterogeneity in CM survivors, which is necessary to improve the precision of our interventions. Further, this project will create an integrative CM dataset that will be a shared data resource for the field, resulting in exponential contributions that extend beyond this K01. Finally, this proposal will greatly enhance the PI’s career development and enable him to advance toward his long-term goal of becoming an independent investigator who can advance the fields of child development and CM via innovative methods. Training-mentorship will be provided to learn IDA methodologies; gain expertise to study risk heterogeneity; acquire skills in longitudinal data analysis; and gain team science skills.

NIH Research Projects · FY 2025 · 2023-09

Asthma is one of the most prevalent chronic diseases of childhood, and a leading cause of pediatric emergency department (ED) visits in the US. Guideline-based asthma management, including treatment with daily preventive medications, can reduce severe asthma flare-ups requiring acute healthcare services. Unfortunately, many children in the ED with asthma are not adequately treated with preventive medications. Poor rates of primary care follow-up after ED discharge result in missed opportunities to optimize treatment and home management and reduce preventable morbidity. Our prior work has demonstrated that a school-based telemedicine intervention for children 3-12 years can facilitate follow-up assessments by primary care providers after ED discharge, and lead to increased prescriptions for preventive medications. Despite access to appropriate treatment, however, the intervention did not lead to improved adherence or reduce symptoms and repeat ED visits. Reducing morbidity for children with asthma in the ED will require a comprehensive guideline-based approach that combines primary care follow-up and treatment with preventive medications, with patient-centered education to support home management and adherence. To enhance home management, we also successfully piloted an intervention of hospital-to-home educational support for children (5-13 years) admitted with asthma that features pictorial materials, health literacy techniques, and a pair of in-home telehealth visits for educational reinforcement after discharge. In response to NOT-OD-21-100: Improving Patient Adherence to Treatment and Prevention Regimens to Promote Health (PA-20-183), we now propose a 2-group randomized trial to test the TEACH-ER (Telehealth-Enhanced Asthma Care for Home after the Emergency Room) intervention vs an enhanced care (EC) control group. The TEACH-ER intervention combines and expands upon our prior work, and includes: 1) brief initial teaching in the ED; 2) in-home telehealth follow-up visit with primary care providers, with a prompted recommendation for guideline-based preventive treatment and home delivery of prescriptions with a pictorial action plan; 3) two additional in-home telehealth visits to deliver health literacy-informed asthma education and home management support. We will enroll 430 children (ages 3-12 yrs) from the two dedicated pediatric EDs in our region. We will assess the effectiveness of the program in reducing morbidity (e.g. repeat ED visits or hospitalizations for asthma, symptom severity, quality of life), improving adherence, and improving preventive asthma care. Participants will complete telephone follow-up assessments 3, 6, 9, and 12 months after enrollment; we will also collective objective data on acute care use from the electronic medical record and prescription fill data. At the study’s completion, the effectiveness of this novel system of care will be better defined as a sustainable means to improve preventive care and reduce morbidity for children with asthma.

NIH Research Projects · FY 2025 · 2023-09

Abstract Platelet parent cells are megakaryocytes (Mks). Studies have historically focused on Mk derived platelet production in the bone marrow (BM) (1). More recently, platelet-producing Mks have been shown to be present in the lung, and our lab and others have described an immune modulatory phenotype of lung Mks (2-4). The recent identification of a low ploidy, immune differentiated BM Mk sub-population (5) highlights the relationship that lower Mk ploidy may correlate with a more immune differentiated Mk. We refer to the development of a Mk immune modulatory sub-population as Mk immune differentiation. The origin of lung Mks and mechanisms of their immune differentiation remain unknown. It is assumed, without experimental evidence, that lung Mks are ‘seeded’ from immature circulating BM Mks. Lefrancais et al. showed the presence of GFP+ Mks in the lung vasculature when a mTmG lung was transplanted to a PF4-Cre-mTmG recipient. The authors argued that the presence of GFP+ intravascular lung Mks demonstrated an extra-pulmonary source. However, intravascular and extravascular lung Mks have morphological and perhaps functional differences, and extravascular lung Mks make up about 70% of all lung Mks. Thus, the majority of circulating Mks would have to leave the blood and tissue migrate to become lung resident. Without direct evidence for this trafficking, the definitive origin of extravascular lung Mks remains unknown, but may have a major impact on their immune differentiation. I hypothesize that extravascular lung Mks derive from a local hematopoietic progenitor pool independent of BM megakaryopoiesis. I will test this hypothesis by using complementary methods, including irradiation and transfer experiments, lineage tracing, and in vitro colony forming unit assays. The impact of Mk immune differentiation on the platelet pool is still not clear. Lung Mks make platelets, although the relative proportion of lung derived platelets remains controversial (4, 6). Emerging evidence from studies of platelet heterogeneity are beginning to suggest that platelet subsets may have functional significance. I hypothesize that lung resident Mks contribute to an immune-differentiated platelet sub-population. To test this hypothesis, I will use a novel strategy to identify lung derived platelets using oropharyngeal CFSE administration in physiologic and thrombocytopenic conditions such as PF4-Cre-iDTR mice and nonlethal murine malaria. Results from my proposed studies will contribute to an improved understanding of immune differentiated megakaryopoiesis, and its role in coordinated immune responses and platelet immune phenotypes, with direct applications to many vascular inflammatory diseases.

NIH Research Projects · FY 2024 · 2023-09

Persons living with HIV/AIDS (PLWHA) are understudied and underserved with respect to both dental and mental health services, compared to the general population. Psychiatric and dental comorbidities in PLWHA result in diagnostic complexities, treatment gaps, increased health care costs and poorer outcomes. Early diagnosis and treatment access are imperative. There are no US studies of co-occurring oral diseases and mental illnesses in PLWHA. Assessment of treatment needs, self-management, and case management can inform improved referral and treatment outcomes. Studies have identified barriers to dental care in PLWHA, including ability to pay, dental anxiety, transportation, etc. Patients, HIV program directors, case managers and administrators are key stakeholders in understanding PLWHA use of dental and mental health services. Their referral success has not been assessed through traditional HIV surveillance. Factors for successful referral of PLWHA to mental health and dental services are understudied. Examining linkage factors as perceived by PLWHA, program directors and other staff, including the role of motivational Self-Determination Theory-based (SDT) mechanisms is needed. The proposed study will examine factors related to referrals of PLWHA to dental and mental health services and to describe the prevalence of comorbidities in 105 PLWHA, their self- management, and their perception of case management. All patients (with or without co-morbidities) will be assessed. The primary study outcome is keeping dental and/or mental health referral appointments. AIM 1: To examine referral linkage factors (social, biological, or behavioral) in those screened for 1) oral diseases only, 2) mental illness only, 3) both oral diseases and mental illness, or 4) neither (comparison group). Other assessments include 8 key informant interviews (KIIs), 2 focus groups (FGs) with 8-12 participants each and validated surveys. AIM 2: 2a) To describe the prevalence of comorbidities in a sample of PLWHA, the use of self-management strategies and their referral need based on screening and clinical data (periodontal inflammation via salivary biomarker IL-6 at baseline); 2b) To assess feasibility and obtain pilot data to inform a future motivation-based intervention targeting oral and mental health behavior in PLWHA. AIM 3: To examine health status at six months via patient screenings, clinical examination with salivary biomarkers, and a survey. Hypothesis: PLWHA screening positive for dental/mental comorbidities will keep fewer referral appointments than those not screening positive. This could be associated with SDT motivational indices for referral and to linkage factors. This innovative mixed-methods study will inform a needed evidence-base for patients, dental and mental health professionals and other stakeholders, as well as future studies (e.g., a randomized clinical trial) and potential interventions such as Motivational Interviewing strategies to maximize successful referrals.

NIH Research Projects · FY 2025 · 2023-09

Proposal Summary/Abstract Communication access for deaf communities promotes equity and inclusion, thus there is heightened concern regarding the many variables that impact the physical and mental health of sign language interpreters. The Model of Interpreter Injury introduced sign production and style, the interpreting situation, and the interpreter’s individual characteristics as factors associated with injury development. Trademarked by the National Institute of Occupational Safety and Health (NIOSH) in 2011, Total Worker Health (TWH®) combines health protection with health promotion to prevent worker injury and advance well-being, making it a promising holistic approach to address the multivariate factors of interpreter injury. Using mixed-methods and a clinical efficacy trial design, the overall objective of this study will employ implementation strategies to adapt and evaluate a previous integrated TWH® program and pilot test its efficacy on interpreter well-being. My central hypothesis is that interpreters in the health and well-being curriculum (intervention) will demonstrate significantly improved well-being compared to those in a general health program (control). The goal of Aim 1 is to adapt and tailor a previous integrated TWH® program into a health and well-being curriculum for interpreters. Group model building will be used for curriculum development during virtual listening sessions with interpreters and the Framework for Reporting Adaptations and Modifications-Expanded will guide documentation of the process for adapting to the context of interpreting. The goal of Aim 2 is to evaluate implementation outcomes of the curriculum via a collective post-intervention satisfaction survey. Finally, the goal of Aim 3 is to determine the efficacy of the health and well-being curriculum via the NIOSH Worker Well-being Questionnaire (WellBQ) collected pre- and immediately post-intervention, as well as, at 6- and 12-month timepoints in a parallel group randomized control trial. The contribution of the proposed research will effectively implement a health and well-being curriculum to protect and promote the occupational health of interpreters. This project is innovative because it will implement the integrated TWH® program to the contextual needs of interpreters and bring experts in the interpreting field together for the first time to deliver a combined interventional approach. This work relates to the NIOSH priority goal seven for extramural research (promote safe and healthy work design and well-being), along with goals four (reduce occupational musculoskeletal disorders) and six (improve workplace safety to reduce traumatic injuries). Evidence from this Mentored Research Scientist Development Award (K01) will establish me as an independent occupational safety and health researcher, contribute to the body of work dedicated to TWH®, and further inform WellBQ development. I have assembled an outstanding advisory team that will provide me with the necessary training to ensure I develop expertise in three primary domains: 1) dissemination and implementation science, 2) occupational health, and 3) advanced statistics.

NIH Research Projects · FY 2025 · 2023-09

Environmental toxicants and, more specifically, exposure to ambient particulate matter (PM) air pollution increases the risk of developing Alzheimer's Disease (AD) and AD-Related Dementias (ADRD). Yet, our current understanding of the mechanisms by which PM exposure augments the progression of ADRD-related pathology and cognitive impairment is very limited. We posit that airborne ultrafine particles (UFP, <100 nm diameter) are causally related to the development or progression of AD/ADRD. The glymphatic pathway is emerging as a key to maintaining brain health and its dysfunction is implicated in several neurological disorders. This glial- dependent clearance pathway is dedicated to draining soluble waste proteins and its existence has been documented in the brain of multiple species, including humans. The proposal is based on preliminary studies showing that exposure to model UFP significantly suppresses glymphatic fluid transport and increases the β amyloid load in a murine model of AD pathology. We hypothesize that airborne UFP are transported to the brain upon inhalation exposure and cause both systemic and neuroinflammation, thus either indirectly or directly impairing glymphatic fluid flow and accelerating AD/ADRD-like pathology and behavioral deficits in a mouse model of AD. The proposed aims will test our hypothesis by addressing the following questions: Aim 1: How does short (3 days) or repeated (3 months intermittent) exposure to UFP affect glymphatic fluid transport and cognitive performance in young (3 months) and aged (15 months) wildtype mice? Aim 2: Can glymphatic impairment resulting from UFP exposure change the progression of Aβ deposition in a murine AD/ADRD model? Aim 3: Will UFP accumulate and interact with cells along the major glymphatic fluid transport segments? We will here use high-resolution analytical scanning transmission electron microscopy to analyze precisely where in the brain parenchyma the UFP accumulate and interact. Aim 4: We will explore the molecular mechanisms of UFP exposure-related glymphatic impairment and pathological progression in a murine AD/ADRD model via pharmacological inhibition of adrenergic signaling. Moreover, a detailed study of AQP4 vascular polarization response to air particulate matter exposure, sleep disruption and how do adrenergic inhibition reverse these signatures of pathology, would also be explored? The innovative aspects of the proposal build upon a unique multidisciplinary approach where expertise in particulate matter toxicology (Elder), basic and applied chemistry with particular focus on the study of UFP in living systems (Graham), neurobiology of diseases and regenerative mechanisms (Hussain), and fundamentals of waste products and metabolites clearance (Nedergaard), will be combined to address the question if and how do UFP enter, distribute, accumulate, and ultimately undergo bioprocessing and efflux from the brain. The proposed experiments represent the first fundamental analysis and mechanistic underpinning of the interconnection between PM exposure, glymphatics, inflammation, and ADRD initiation and progression.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Retinal ganglion cells (RGCs) provide the sole source of visual information to the brain and form the building blocks for all downstream vision. In primates, considerable progress has been made in characterizing the three most common RGC types, which make up 80% of the retinal output, and much less is known about the remaining 15+ rarer RGC types. A key barrier to progress has been the difficulty of targeting these rare RGCs in acute experiments. These challenges have been overcome with an approach for visualizing the structure and function of foveal RGCs in the living macaque eye by combining calcium imaging, retrograde tracers and fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO). FAOSLO imaging is non-invasive and enables study of the same RGC populations for months or years. This technique has enabled in vivo classification of foveal RGCs to identify the elusive rarer types. This proposal aims to extend the capabilities of FAOSLO imaging to directly test the roles of these rare RGCs in vision while establishing the foundation for an independent research career. Aim One will implement high-speed scanning strategies and voltage indicators to read the retinal code in the living eye and achieve the temporal resolution necessary to study rare motion-sensitive RGCs. Aim Two will directly test the hypothesis that the rare ON direction selective RGC type contributes to optokinetic eye movements in primates using targeted laser lesions of individual RGCs. Aim Three will establish a paradigm to isolate rare RGCs and the visual functions they mediate through transneuronal retrograde degeneration following V1 lesions. This line of investigation will also clarify the timeline of RGC loss and the underlying physiological changes that occur in RGCs following V1 damage in strokes. The research goals of this proposal are reinforced by a comprehensive training plan that will provide the new skills and knowledge necessary to achieve the candidate’s research goal of establishing the links between rare primate RGCs and visual functions. The candidate will carry out the mentored phase with Dr. David Williams, a pioneer in the use of adaptive optics for imaging the eye. Co-mentor Dr. Bill Merigan will contribute expertise in behavioral experiments, lesions and viral vectors. Additional training from a first-rate advisory committee (Drs. Krystel Huxlin, Tony Movshon and Jesse Schallek) will put the candidate on a strong pathway to independence. Together, the research and training proposed will facilitate the candidate’s successful transition to a tenure-track faculty position at a research- intensive university.

NIH Research Projects · FY 2025 · 2023-09

Social Connection & Suicide Risk in ADRD Caregivers Providing care for a family member or friend with Alzheimer’s Disease and Related Dementias (ADRD, ‘caregivers’) is a common experience for adults in mid- and later-life and can foster experiences that are beneficial to mental health (e.g., purpose, mastery), but can also lead to anxiety, depression, and suicide ideation. Our scientific premise is that social disconnection—the quality and quantity of social ties with others— contributes to the most severe mental health problems during caregiving, including suicide ideation and behavior. Social disconnection is a common experience among caregivers and is one of the strongest risk factors for suicide ideation, attempts, and deaths. The Interpersonal Theory of Suicide proposes that stressors such as caregiving increase suicide risk by impacting two forms of social disconnection—feeling lonely (also termed low belonging) and like one is a burden on others (perceived burden). In line with NIA priorities of promoting well-being in caregivers and understanding mechanisms, our objective is to examine loneliness (and low belonging) and perceived burden on others as mechanisms that increase suicide risk in ADRD caregivers. This study builds on our team’s work on social connection in ADRD caregivers via the Rochester Roybal Center for Social Ties & Aging Research. Our pilot studies routinely assess suicide risk given the robust link between social disconnection and suicide risk. We found that at least 10% of caregivers screened for our pilot studies report suicide ideation in the past month. We propose an observational study of n=200 ADRD caregivers who report caregiving stress, loneliness, and recent suicide ideation in which subjects complete baseline interviews (to characterize caregiving and obtain histories of suicide ideation and behavior), 10 days of smartphone-based ecological momentary assessment (daily caregiving stressors and benefits, social connection, and suicide ideation), and 6-month follow-up (changes in care receiver functional status). Our first aim is to examine whether daily caregiving experiences are associated with daily variations in loneliness and perceived burden. We hypothesize that caregiving mastery and sense of purpose in life will be associated with lower loneliness and greater perceived burden, while caregiving strain, losses, and relationship conflict will be associated with greater loneliness and perceived burden. Our second aim examines whether loneliness and perceived burden predict daily variation in suicide ideation. We hypothesize that caregiving stressors will predict suicide ideation via increases in loneliness and perceived burden. Our third aim is to examine whether suicide ideation in caregivers is associated with worse caregiving outcomes in the short-term (next day caregiving stress, fewer positive interactions) and longer-term (functional decline at 6-month follow-up). Understanding mechanisms whereby caregiving may increase suicide risk could identify social connection as an intervention target and also increase knowledge about the role of daily stressors in the link between social disconnection and suicide in mid- and later life.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Perfluorohexanoic acid (PFHxA) is a short-chain per- and polyfluoroalkyl substance (PFAS) used in industrial and consumer products, such as aqueous film forming foam (AFFF), food wrappers, stain repellants on furniture, and makeup. PFHxA use is not regulated in the United States. PFHxA is found ubiquitously in the environment significantly contaminating soil and water resulting in humans being exposed through ingestion. Epidemiology literature has reported PFHxA in maternal serum and breastmilk, and PFHxA crosses the placenta. Together, these data demonstrate that fetuses are exposed through the placenta and infants through breast feeding. In humans, PFHxA accumulates higher in the cerebellum than in most other brain regions. The cerebellum is critical to both motor and cognitive functions, is the home of 50% of the neurons, has a unique cytoarchitecture, and develops later than other regions making it an important region to study in the context of developmental toxicant exposures. While the health effects of PFHxA are still largely unstudied, it is well established that legacy PFAS alter immune function, including suppressing peripheral immune responses. However, the effects of PFHxA on the immune cells of the brain, microglia, have not been investigated. Microglia have the same mesodermal origins as peripheral immune cells, suggesting that developmental exposures may target microglial function. Interestingly, microglia in the cerebellum may be particularly sensitive to neurotoxicants compared to microglia in other brain regions as they have a unique transcriptomic profile. In addition to immune function, microglia play a critical role in brain development by aiding in angiogenesis, providing factors for myelin development, and pruning synapses thereby remodeling neural circuits. Thus developmental PFHxA exposure could affect their immune and neuroanatomical functions. Despite the importance of microglia in brain development and evidence that PFHxA enters the brain, PFHxA has not been investigated in the mammalian nervous system. Based on evidence from epidemiology studies on PFHxA as well as long-chain PFAS, I hypothesize that gestational and lactational exposure to PFHxA in a mouse model disrupts cerebellar development, affecting neuronal and glial phenotypes, as well as motor activity. Microglia may be uniquely affected by this exposure, becoming immunosuppressed. To test this hypothesis, I will pursue two aims using a mouse model representing human exposure to PFHxA during gestation and lactation. The first aim will investigate how gestational and lactational exposure to PFHxA affects neuron and glia phenotypes using RNA sequencing and immunohistochemistry (IHC). This aim will also investigate how this exposure affects animal behaviors, especially those related to the cerebellum. The second will determine if microglia are altered at the genetic, protein, and dynamic level by using RNA sequencing, IHC, and two-photon microscopy. These experiments will show, for the first time, whether PFHxA exposure during brain development is neurotoxic and will also guide regulators from multiple agencies when considering PFHxA exposure to humans.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Horizontal gene transfer (HGT), specifically plasmid conjugation, is a driving force in microbial evolution and pathogenesis. The process of conjugation appears deceptively simple: a donor cell transfers a copy of a plasmid to a compatible recipient cell through a physical mating bridge. In doing so, diverse traits, such as metabolic, virulence, and antibiotic resistance genes, can be spread. As such, HGT has been implicated in a variety of human health and industrial applications, ranging from multi-drug resistance to bioremediation. Advances in microbiome studies have revealed that HGT occurs between both closely and distantly related strains, yielding a wide diversity of potential strain/plasmid combinations; despite this, epidemiological surveillance clearly demonstrates that only a small minority of clones and their associated plasmids persist in situ and are highly conserved across different ecological, geographical, and clinical contexts. Thus, it is widely believed that the overall fitness of individual strain-plasmid pairs is a key feature of successful pathogens. Fundamentally, this success is driven by a dynamic interaction between a plasmid-carrying donor and suitable recipient strain in a favorable environment, resulting in the formation of new strain-plasmid pairs (e.g., transconjugants). However, research to date has primarily focused on established strain-plasmid combinations (e.g., donor capabilities and/or plasmid fitness costs); in contrast, the dynamics and factors favoring the initial formation of these combinations are entirely unknown. Yet, such information is critical to both predict new pathogen emergence and develop strategies that intervene in plasmid acquisition before they become established in a population. To address this gap, my research program leverages our unique interdisciplinary expertise in computational modeling, bioinformatics, and mechanistic experiments to investigate the molecular factors favoring the formation of new strain-plasmid combinations. Our proposed themes approach this problem from three complementary perspectives: (1) What genetic features make certain plasmids harder/easier to acquire? (2) What determines a strain’s potential to act as a good HGT recipient? (3) How does environmental selection impact plasmid acquisition capabilities? Combined, these parallel objectives work towards a unified framework that integrates insights across multiple levels of complexity (i.e., molecular to ecological/evolutionary). These research directions contribute to our long-term goal, one that is central to the NIGMS mission, of reliably predicting (and ultimately controlling) clinically relevant strain/plasmid prevalence, and will eventually enable us to anticipate pathogen emergence a priori and explore downstream applications, e.g., novel antibiotic treatment strategies.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Whereas peanut (PN) allergy is the most common cause of fatal and near-fatal food-allergic reactions in older children and adults, egg and cow’s milk are the most common food triggers of anaphylaxis in infancy. Egg allergy is the most common childhood IgE-mediated food allergy with prevalence rates ranging from 1.3-1.6% in general population up to 20% in high-risk infants. Egg sensitization is a significant risk factor for PN allergy and early aeroallergen sensitization, which can lead to the “atopic march”. Black children are over-represented among children with egg allergy and asthma is more prevalent in children with allergy to egg than any other food. Only 50% develop tolerance to egg by school-age; PN allergy is rarely outgrown. There is a critical “window of opportunity” for the introduction of allergenic solids around 4-6 months in decreasing the risk of food allergy as shown in randomized controlled trials with egg and PN. However, 36% of infants were already sensitized to egg at enrollment and about 9% to PN, excluding them from early introduction; 20% infants were clinically allergic, even anaphylactic, on their first introduction of egg. To be more effective, primary prevention strategies must begin much earlier before sensitization is established, possibly even during pregnancy or breastfeeding. Prior studies are conflicting in the effect of maternal diet including or excluding egg or PN on development of food allergy in infancy. Per the American Academy of Pediatrics, “Current evidence does not support a role for maternal dietary restrictions during pregnancy or lactation”, but randomized controlled trials are lacking. Thus, there is an urgent need to provide mothers with concrete advice on feeding practices based on good quality evidence. The overall objective of this application is to define the role for egg and PN exposure during pregnancy and via human milk in food sensitization and to identify the biomarkers of sensitization or tolerance. We will conduct a nationally-enrolled, randomized controlled, four-arm parallel trial. Mothers pregnant with infants at high risk (with first degree relative with an atopic disease) who intend to breastfeed >3 months will be enrolled utilizing social media and referrals and randomized to either consumption or avoidance of egg and PN containing foods throughout pregnancy and lactation to assess the risk of sensitization to egg and PN at 4 months before introduction of solid foods. In Aim 1, we will determine the effect of maternal dietary egg and PN avoidance during pregnancy and lactation on the risk of egg and PN sensitization in high-risk infants. In Aim 2, we will assess the level of infant oral and environmental exposure to egg and PN. In Aim 3, we will identify the immune responses elicited by maternal diet including or excluding egg and PN during pregnancy and lactation. Knowledge gained from these studies will uncover mechanisms of early-life sensitization necessary to establish maternal dietary guidelines in prevention of food allergy and the atopic march. 1

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Retinal ganglion cells (RGCs) provide the sole source of visual information to the brain and form the building blocks for all downstream vision. In primates, considerable progress has been made in characterizing the three most common RGC types, which make up 80% of the retinal output, and much less is known about the remaining 15+ rarer RGC types. A key barrier to progress has been the difficulty of targeting these rare RGCs in acute experiments. These challenges have been overcome with an approach for visualizing the structure and function of foveal RGCs in the living macaque eye by combining calcium imaging, retrograde tracers and fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO). FAOSLO imaging is non-invasive and enables study of the same RGC populations for months or years. This technique has enabled in vivo classification of foveal RGCs to identify the elusive rarer types. This proposal aims to extend the capabilities of FAOSLO imaging to directly test the roles of these rare RGCs in vision while establishing the foundation for an independent research career. Aim One will implement high-speed scanning strategies and voltage indicators to read the retinal code in the living eye and achieve the temporal resolution necessary to study rare motion-sensitive RGCs. Aim Two will directly test the hypothesis that the rare ON direction selective RGC type contributes to optokinetic eye movements in primates using targeted laser lesions of individual RGCs. Aim Three will establish a paradigm to isolate rare RGCs and the visual functions they mediate through transneuronal retrograde degeneration following V1 lesions. This line of investigation will also clarify the timeline of RGC loss and the underlying physiological changes that occur in RGCs following V1 damage in strokes. The research goals of this proposal are reinforced by a comprehensive training plan that will provide the new skills and knowledge necessary to achieve the candidate’s research goal of establishing the links between rare primate RGCs and visual functions. The candidate will carry out the mentored phase with Dr. David Williams, a pioneer in the use of adaptive optics for imaging the eye. Co-mentor Dr. Bill Merigan will contribute expertise in behavioral experiments, lesions and viral vectors. Additional training from a first-rate advisory committee (Drs. Krystel Huxlin, Tony Movshon and Jesse Schallek) will put the candidate on a strong pathway to independence. Together, the research and training proposed will facilitate the candidate’s successful transition to a tenure-track faculty position at a research- intensive university.