Yale University

NIH Research Projects · FY 2026 · 2023-12

Vascular smooth muscle cells (VSMCs) have a dramatic ability to alter their phenotype to adopt different roles in diverse tissues and organs and contribute to vascular physiology, growth, remodeling, and repair. The inappropriate SMC phenotypic modulation, however, results in a number of cardiovascular pathologies, including aortic aneurysm, atherosclerosis, vascular malformation, systemic and pulmonary hypertension. To this date, there has been no cure for these conditions, and surgical repair or transcatheter interventions are often associated with the recurrence of the disease or major complications. PRDM6 is an SMC-specific epigenetic regulator that is most abundant in vascular smooth muscle cells (VSMCs) and regulates its plasticity. Genetic variants in the PRDM6 gene have been associated with a wide range of traits, including blood pressure regulation, reduced thoracic aortic distensibility and aortic dilation, intracranial aneurysm, coronary artery disease, type II diabetes (T2D), BMI, and atrial flutter by genome-wide association studies (GWAS). How PRDM6 is transcriptionally regulated and the epigenetic mechanisms by which it regulates gene transcription are not known. Our goal is to outline the regulatory landscape of PRDM6 in the human thoracic aorta, and identify PRDM6 enhancers that by regulating PRDM6 transcription determine the aortic diameter by combining high throughput reporter assays with genome-wide association data. Further, we will delineate the mechanisms of gene regulation by PRDM6 in mouse aortic SMCs (ASMCs) by genome-wide ChIPseq assays and identify PRDM6-regulated genes in peak GWAS loci for cardiovascular diseases. Finally, we will examine the role of Wnt signaling in aortic dilation and examine the effects of its inhibition in rescuing the trait. These findings are expected to lead to target identification for the development of novel therapeutics for aortic aneurysms and other diseases arising from dysregulated VSMCs, including aortic coarctations, and systemic hypertension.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Our long-term goal is to understand the cellular and genetic rules governing how endothelial cells (EC), vascular smooth muscle cells (VSMC) and sympathetic neurons (SN) interact within the artery. SNs innervate vascular SMCs and control normal vascular functions such as circulation, respiration, body temperature, and metabolism. Thus, aberrant regulation of the vascular-mural-sympathetic (EC-VSMC-SN) interaction underlies numerous cardiovascular pathologies which affect millions of people worldwide. Although much progress has been made in identifying the molecular signaling controlling the individual EC, VSMC, and SN vascular functions, less is known about the players that allow the three cell types to work as a unit. This lack of knowledge is underscored by the limited strategies available to improve vascular function by manipulating the sympathetic vascular unit. Here we will use a basic science approach to address this problem through experiments designed to unravel the cellular and genetic mechanisms that govern normal EC-VSMC-SN unit formation. We will first understand, via live-cell imaging microscopy, how blood flow coordinate EC-VSMC-SN unit assembly at the dorsal aorta in zebrafish (Aim 1) and then dissect the function of the identified flow-depended genetic programs in each cells to allow the neurovascular coupling (Aim 2). Finally, in Aim 3, thanks to our successful collaboration with Drs. Anne Eichmann, Martin Schwartz and Martina Brueckner, the signaling and cellular study of the EC- VSMC-SN unit will be translated in the mammalian cardiovascular system. Given the therapeutic potential of manipulating sympathetic innervation, our studies will have a broad, positive impact on identifying novel targetable signaling pathways to control sympathetic function.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Respiratory syncytial virus (RSV) is a significant source of morbidity and mortality in the pediatric population. It is estimated that every year, over 100,000 children under 5 years of age die from RSV, half of whom are infants <6 months of age. The burden that RSV exerts on healthcare systems cannot be overstated. In the United States, RSV is associated with 1.5 million annual medical encounters in children less than five years and is the leading cause of hospitalization among infants under one year of age. The pending introduction of new immunoprophylactic agents offers unique opportunities to confront the challenge of RSV in infants. Two products whose approval is imminent include a vaccine that is administered to pregnant women to protect their newborns and a long-acting monoclonal antibody (mAb) that is deployed like a vaccine and routinely given to all newborns. As is the case with any new vaccine, it will be important to conduct studies during the early phases of implementing these new immunization strategies to answer many unanswered questions surrounding their risks and benefits in real-world settings. To address this critical need, we propose a large- scale case-control study that integrates data from different modalities (e.g., clinical, demographic, virologic, immunologic) to evaluate the effectiveness of perinatal RSV immunoprophylaxis. Cases and controls will be identified using active surveillance in both inpatient and outpatient clinical sites of the Yale New Haven Health System, the largest and most comprehensive healthcare system in Connecticut, with 8 large hospitals and close to 4 million outpatient encounters every year. We will enroll patients seeking health care for acute respiratory illness and confirm RSV infection using approved molecular assays. We will use data-gathering protocols that are both exhaustive and identical for both cases and controls and collect data from multiple sources, including health records, interviews, immunization registries, and population-based surveys. We will generate estimates of effectiveness for each type of immunization used, disaggregated by time from immunization, disease severity, and sociodemographic characteristics (Aim 1). We will also perform genetic characterization of all the study's RSV cases, monitor the virus's genetic diversity over time, and quantify the relative effectiveness of immunoprophylaxis against various viral lineages (Aim 2). Finally, we will collect acute and convalescent blood from a subset of infants and employ a single-cell and multi-omics approach to study the dynamics of the innate and adaptive immune responses during RSV infection and explore the molecular mechanisms that contribute to immunoprophylaxis failure (Aim 3). The results from this project will be critical as it will allow policymakers to determine whether these products are sufficiently beneficial to warrant continual inclusion in the immunization program. Further, if our results show that immunization confers considerable protection, these data can be a potent incentive to increase the strength of recommendations by healthcare providers and boost acceptance by patients.

NIH Research Projects · FY 2026 · 2023-12

Project Summary CDKL5 Deficiency Disorder (CDD) is an X-linked genetic disorder caused by mutations in the cdkl5 (cyclin-dependent kinase-like 5) gene. Patients with CDD exhibit many features common to Autism Spectrum Disorder, but also frequently suffer from cortical visual impairment. Specifically, patients with CDD display deficits in visually-evoked potentials, an indirect measure of visually evoked thalamocortical input. Emerging evidence suggests that loss of CDKL5 is also associated with neuronal hyperexcitability. Together, this suggests that loss of CDKL5 may shift the balance between thalamocortical and intracortical processing. Mice lacking CDKL5 recapitulate many aspects of human CDD patients, including visual impairment, and also exhibit changes in arousal. Work from our lab and others has demonstrated that arousal states reflect distinct underlying physiological processes that influence spontaneous and sensory-evoked neural activity. However, the relative impact of behavioral state dysregulation on thalamocortical dynamics and the contribution of a shift from feedforward inputs to intracortical network control on visual processing remains unknown. Leveraging a recently developed technique from our lab, we can simultaneously monitor spontaneous and visually-evoked thalamic axon activity in the primary visual cortex and cortical activity across the dorsal neocortex in the awake, behaving mouse. Using this approach, we have begun to characterize the functional consequences of CDKL5 deficiency on state-dependent thalamocortical dynamics and visual processing. Our preliminary data demonstrates that CDKL5 deficient mice display altered arousal states and cortical activity in a sex-dependent manner, along with increased intracortical functional connectivity. Initial experiments suggest that CDKL5 deficient mice also exhibit reduced visual response magnitudes in in primary visual cortex. We will therefore test the following hypotheses: 1) dysregulation of behavioral state in CDKL5 deficiency also disrupts thalamocortical network activity; 2) loss of CDKL5 results in an increase in intracortical connectivity and a decrease in thalamocortical connectivity; 3) CDKL5 deficiency leads to reduced thalamocortical visual sensitivity; and 4) CDKL5 deficiency decreases feedforward transformation of sensory information. Together, our results will provide unprecedented insight into the functional consequences of CDKL5 deficiency for state-dependent thalamocortical networks and visual processing in a mouse model of CDD.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Thoracic aortic aneurysm (TAA) is a degenerative aortopathy that affects children and adults and predisposes to sudden thoracic aortic dissection. Patients with TAA require lifelong cardiac care intended to prevent progressive dilation or dissection. Cardiac decision-making requires projection of future risk, but the status quo for establishing prognosis is not personalized and lacks precision. The basis for rapid progression versus stability or improvement over time is not understood nor predictable using current approaches. Novel methods to classify risk for disease progression upon diagnosis are urgently needed. Our long-term objective is to leverage molecular and image analysis tools to create novel methods to classify risk of aortopathy progression precisely and identify mechanisms amenable to future therapeutic targeting. Our first aim is to identify genetic variants that are associated with increased or decreased longitudinal rate of aortic dilation, hypothesizing that single nucleotide polymorphisms (SNPs) regulate inter-individual variability in TAA progression. We will perform whole genome sequencing (WGS) in study participants who are receiving longitudinal pediatric aortopathy care. Enrollment will occur at two pediatric cardiac centers with subspecialty aortopathy programs: Indiana University Health’s Riley Hospital and Children’s Healthcare of Atlanta. We will test SNPs for their association with rates of aortic dilation using mixed model analysis of serial aortic diameter measurements. In our second aim, we will leverage our unique aortopathy tissue biobank to identify novel candidate SNPs in transcriptional regulatory elements that contribute to TAA pathogenesis. We will perform deep RNA sequencing of flash frozen human TAA tissues and controls and integrate these data with WGS to identify genes with differential allele-specific expression in TAA. We will directly test the impacts of candidate SNPs in 3’-untranslated regions and enhancers on allelic transcription using our high-throughput reporter assays in cultured human aortic smooth muscle cells. For our third aim, in collaboration with biomedical engineers at Purdue University, we have pioneered a novel automated algorithm that tracks aortic root kinematics across cardiac cycles and extracts morphological and functional metrics using clinical echocardiography. We will establish a large normative data set of novel metrics in young healthy subjects and compare metrics between TAA cases and matched controls. We hypothesize that abnormal metrics portend increased rate of aortic dilation at follow-up, helping us predict clinical outcomes. Upon completion, these aims will elucidate a genetic basis for transcriptional dysregulation in TAA, identify SNPs that modify TAA progression, and advance echocardiographic phenotyping of the aortic root. Creation of a novel classifier to predict risk for TAA progression, without significantly modifying existing clinical workflows, is a major expected outcome. Our biobank’s depth will enable further mechanistic investigation of the SNPs identified in this study. Automated aortic root phenotyping could transform aortopathy evaluation and propel clinical research.

NIH Research Projects · FY 2025 · 2023-12

7. Project Summary/Abstract Complement may be activated in the peri-transplant period by insults such as ischemia reperfusion injury or binding of preformed host-anti-graft antibodies as well as post-transplant by binding of de novo donor specific antibody. In these settings, complement membrane attack complexes (MACs) are deposited on allograft endothelial cells (ECs) which increases the capacity of graft ECs to activate alloreactive host T and NK cells, providing a mechanistic link between such insults and an increased incidence and severity of T cell-mediated rejection (TCMR) without overt EC damage. We have shown that MACs are internalized and initiate IL-1 synthesis, processing and secretion followed by IL-1-mediated autocrine/paracrine activation of human ECs. A key component of this activation response is expression of IL-15/IL-15R complexes on the EC surface that can be trans-presented to circulating lymphocytes. Blocking IL-15 trans-presentation reverses much of the MAC-induced augmentation of EC alloimmunogenicity. We hypothesize that gene editing of graft ECs ex vivo prior to transplant will reduce the severity of TCMR and prolong graft survival while still enabling the host immune system to respond to and control graft infection. Here we propose to develop approaches to do so, but preliminary data have revealed that the response of ECs to MAC and IL-1 is unexpectedly complex. Human ECs transcribe multiple different versions of IL-15 mRNA and increase these transcripts in response to IFN- or MAC/IL-1, but only MAC/IL-1 leads to surface expression. The MAC/IL-1 response requires activation and nuclear uptake of NF-B, but NF-B appears to regulate IL-15 translation rather than transcription, possibly mediated by miRNA. Furthermore, co-induction of IL-15R, rather than pre-existing copies of this protein, is required for endogenous IL-15 surface expression. It is unknown if exogenous IL-15 can be trans-presented by ECs. We need to better understand these pathways in order to exploit them therapeutically. In this R21, we will identify and quantify the IL-15 mRNA species expressed by cultured human ECs that encode surface expressed protein isoforms and elucidate the mechanism of their translational control (aim 1). We then propose to optimize strategies for the elimination of MAC-induced IL-15 surface expression and trans- presentation using CRISPR/Cas9 gene editing and determine if and how this can be achieved using mRNA transfection while preserving EC homeostatic functions (aim 2). We will use the results of these studies to guide our future approach to gene edit human graft EC in ex vivo machine perfused organs.

NIH Research Projects · FY 2026 · 2023-12

Single ventricle congenital heart defects, in which one ventricle fails to develop leading to mixing of oxygenated and deoxygenated blood, affect approximately 1 in 1,000 live births and have a 70% mortality rate. The Fontan operation is the standard surgical treatment, where venous blood is diverted directly to the pulmonary artery via synthetic or tissue-engineered vascular conduits (TEVCs). TEVCs are of great interest and promise due to their ability to remodel and grow with children. However, an unexpectedly high incidence of graft stenosis was reported in a prior TEVC clinical trial, leading to termination. It is likely that overt inflammatory responses caused by graft material degradation and pre-seeded bone marrow cells may have led to over-proliferation of repopulated host cells and graft stenosis. By replacing bone marrow cells with human induced pluripotent cell-derived endothelial cells (hiPSC-ECs) and substituting biodegradable synthetic grafts with native decellularized human umbilical arteries (dHUAs), we generated TEVCs via coating the lumen of dHUAs with hiPSC-ECs under physiological shear stress in a flow bioreactor. TEVCs prevented luminal stenosis and clotting after implantation as inferior vena cava (IVC) interposition grafts in nude rats, a validated model for studying grafts for Fontan procedures. To make TEVCs immunocompatible to any patient, we have generated hypoimmunogenic universal hiPSC-ECs via ablation of human leukocyte antigens (HLAs) using the CRISPR gene editing. The therapeutic efficacy and immunocompatibility of universal TEVCs will be investigated via IVC implantation in immune-humanized rats. Graft patency and blood flow will be monitored by ultrasound, and grafts will be harvested for histological analysis within 3 months post-implantation. Expanding on using universal, endothelialized vascular conduits, the PI’s group will also develop a contractile Fontan conduit as a generation 2 therapy to assist blood flow from IVC to the pulmonary artery, as none of the conduits now in use provide pumping activity, leading to insufficient tissue perfusion, heart failure, and pulmonary vascular disease. We have recently developed a tissue-engineered pulsatile conduit (TEPC) by deriving engineered heart tissues (EHTs) made by seeding hiPSC-derived cardiomyocytes into decellularized native heart matrices and then wrapping the EHTs around a dHUA. We will develop an immunocompatible TEPC by wrapping EHTs based on universal hiPSC-derived cardiomyocytes and cardiac fibroblasts around the above vascular conduits. TEPCs will be matured under electro-mechanical training conditions in bioreactors to achieve enhanced contractile output to make a strong Fontan conduit that assists pulmonary circulation. The PI will test the hypothesis that universal TEPCs are immunocompatible and maintain contractility in the IVC graft model in immune-humanized rats. Ultrasound will be employed to monitor the patency and pulsatility of TEPCs, and TEPCs will be explanted for histological analysis. Universal hiPSC-TEPCs will establish the foundation for generating readily available, mechanically active Fontan conduits, providing a curative therapeutic for patients born with single ventricle congenital heart defects.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT Our focus is identifying molecular signatures defining sickle cell disease and reduced vaccine response by investigating multiple components of innate immunity and the quality and quantity of long-term adaptive immunity among children with sickle cell disease. We propose a deep interrogation and a systems biology approach to characterize changes in innate and adaptive immunity and whole-genome transcriptional response induced by vaccination in a dysregulated inflammatory milieu linked to poor vaccine response. We have assembled an interdisciplinary investigative group who have an existing successful collaboration infrastructure and has expertise in human immunology, pediatric vaccine response, sickle cell disease, pneumococcal antibody response, proteomics, and computational biology to carry out the proposed studies by 1) examining the innate immune status of children with sickle cell disease; 2) elucidating innate and adaptive signatures of vaccination; 3) defining transcriptomic and proteomic signatures of vaccine response. We will leverage the recent advances in single-cell and spatial immune profiling methods and shared immunologic and proteomic platforms to create a novel resource for sickle cell disease patients and other vulnerable populations. We aim to provide comprehensive mechanistic insights into sickle cell disease-related chronic inflammation and impaired vaccine response that will lead to new interventions to improve clinical outcomes in this understudied pediatric population that have increased morbidity and mortality from infectious diseases and impaired responses to vaccination.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Genetic variants in the TRIO gene increase risk for neurodevelopmental disorders (NDDs) including schizophrenia, autism, and related disorders. A cluster of NDD-associated variants selectively impacts TRIO guanine nucleotide exchange factor domain 1 (GEF1) activity, which activates the Rac1 and RhoG GTPases, but the downstream molecular mechanisms by which TRIO GEF1 activity regulates neuronal development and whether they can be targeted therapeutically are not known. We discovered a key autoregulatory mechanism, and showed that NDD-related genetic variants impact this mechanism to enhance or inhibit TRIO GEF1 activity. Here, we propose to employ a novel construct that includes the physiologically-relevant autoregulatory elements in a high throughput screen to discover and validate positive and negative modulators of TRIO GEF1 function. These probes will be critical to advance our understanding of TRIO GEF1 function and its regulation, to probe candidate disease mechanisms, and to potentially suggest novel therapeutic strategies to address a spectrum of NDDs. Aim 1. Identify positive and negative small molecule modulators of TRIO GEF1 activity. The GEF1 domain alone catalyzes robust GTP exchange on Rac1 in vitro, whereas the SR6-GEF1 construct which includes the adjacent spectrin repeats (SRs) 6-9 reduces GEF1 catalytic activity 7-fold. Introducing NDD-related SR variants into SR6-GEF1 increases its activity, while variants in GEF1 domain reduce its activity, indicating that small chemical changes dramatically impact GEF1 activity. We will use a HTS biochemical assay of 150,000 structurally diverse drug- and lead-like compounds to identify those that increase or decrease the SR6-GEF1 activity. Aim 2. Verify and evaluate HTS hits using secondary assays. Hit compounds will be rescreened for confirmation and run in assays lacking SR6-GEF1 to rule out false positives. We will verify hits by repurchasing selected hits as dry powders and retesting their activity against SR6-GEF1. We will measure SR6-GEF1- mediated activation of Rac1 vs. RhoG to determine the substrate specificity of the compounds and investigate isoform selectivity for TRIO GEF1 by testing confirmed hits against a panel of neuronal GEFs. We will test a select group of the most potent hits to address mechanisms of action, including KM and kcat for Rac1 and RhoG, whether compounds bind SR6-GEF1, and how they impact SR6-GEF1 binding to Rac1 and RhoG. Aim 3. Assess probe efficacy on TRIO GEF1 activity in cells and neurons. We will study a set of the most potent and selective activators and inhibitors for their ability to penetrate cells and modulate TRIO GEF1 activity using both a Rac1 biosensor and a quantitative readout of TRIO GEF1 activity on HEK293 cell morphology. We will measure whether a select group of inhibitors recapitulates defects in dendrite development and synaptic transmission resulting from reduced TRIO GEF1 activity. We have also generated CRISPR mice heterozygous for GEF1-defective NDD-related TRIO variants, and will test whether activators can normalize deficits in dendrite development and synaptic function in cultured neurons and brain slices from these mice.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY RNA modifications are chemical changes made to transcripts that can regulate their processing, structure, and stability. Recent advances in modification detection techniques have revealed the presence of RNA modifications in mRNA and lncRNA, expanding the known regulatory potential of these modifications beyond their canonical roles in tRNA and rRNA. RNA modifications have been found to modulate the expression of oncogenes and tumor suppressors alike, demonstrating the need to better understand the basic mechanisms of this process so that specific and effective cancer therapeutics can be developed. A critical gap in the literature is the spatial context of modified transcripts; many studies use RNA from whole cells and may miss key mechanisms by averaging the effects of RNA modifications across the transcriptome. I will bring a new perspective to RNA modification biology by focusing my work on a single subcellular context: the paraspeckle. Paraspeckles are stress-inducible nuclear bodies that are assembled on the lncRNA NEAT1, and both this transcript and the paraspeckle itself have been implicated in human cancers. I have used mass spectrometry and sequencing to identify novel RNA modifications on NEAT1, and I hypothesize that these and other modifications on NEAT1 contribute to the assembly of functional paraspeckles. Critically, my preliminary results indicate that the modification profile of NEAT1 differs between cell lines, so I will perform experiments in lines from two cancers marked by overexpression of NEAT1 and one where NEAT1 is downregulated, so that I can look for common mechanisms as well as patterns in the differences between them. In Aim 1, I will focus on NEAT1 directly. I propose the expansion of my current mass spectrometry and sequencing methods so that I can assemble a more complete map of RNA modifications on NEAT1, including the validation and quantification of modifications at single-base resolution. I will mutate identified modification sites, then measure the effects on NEAT1 stability and isoform distribution by qPCR and effects on protein interactions through crosslinking and proteomic analysis. In Aim 2, I will investigate the paraspeckle. I will use both an unbiased genome-wide screen using a paraspeckle reporter system and a targeted microscopy screen of known RNA modification enzymes to identify novel regulators of the paraspeckle. I will make catalytically inactive mutants of the top hits from these screens and perform modification-sensitive RNA sequencing to determine whether these enzymes are modifying NEAT1, other components of the paraspeckle, or upstream regulators, and use fluorescence recovery after photobleaching to measure modification-specific changes in paraspeckle dynamics. Together, these aims will discover and characterize RNA modifications that have a role in paraspeckle formation, revealing insights in an unexplored area of RNA cell biology.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Emergency myelopoiesis (EM) is a rapid response by hematopoietic stem and progenitor cells (HSPCs) that is activated in response to inflammatory stimuli such as severe infection and cancer, wherein HSPCs proliferate and in increase their output of myeloid cells to replenish bone marrow reserves of those that have been deployed to sites of inflammation. Accumulating evidence has now shown that following the resolution of inflammation, HSPCs can maintain an altered epigenetic program over time. This process, termed “trained immunity” is a type of epigenetic memory that can result in more rapid responses to subsequent inflammatory challenges, such as infection and cancer. Despite growing evidence of its importance in regulating responses to inflammation, the cellular players and molecular pathways involved in controlling the epigenetic responses that lead to trained immunity are poorly understood. In preliminary studies, we performed scRNA-seq in sorted bone marrow immune cells following challenge with β-glucan, a stimulus which induces trained immunity following a rapid EM response. This analysis identified robust activation of bone marrow group 2 innate lymphoid cells (ILC2s) to produce GM-CSF. Blocking either GM-CSF or deleting ILC2s abrogated the EM response β-glucan. However, whether ILC2s or GM-CSF promote trained immunity in this model is not known. Based on this data, we propose that GM-CSF elaborated by ILC2s in response to β-glucan plays a critical role in the establishment of trained immunity. Here we propose to test this hypothesis by (i) defining the role of GM-CSF in trained immunity; (ii) establish the role of ILC2s in this system through an ILC2-deficient mouse line; and (iii) determining the gene expression networks and epigenetic landscapes established by GM-CSF downstream of β-glucan challenge in HSPCs. Altogether, completion of these studies will fundamentally advance our understanding of the regulation of trained immunity. Furthermore, as trained immunity is actively investigated as an intervention in the clinic, modulation of ILC2-GM-CSF pathway could be a novel therapeutic target for the treatment of inflammatory diseases and cancer. Finally, completion of these studies will establish the groundwork for future efforts to identify key cellular and molecular pathways involved in regulating trained immunity in vivo.

NIH Research Projects · FY 2026 · 2023-12

Project Summary: This proposal will interrogate pre-synaptic release properties in vivo in order to extend observations made in reduced system to the awake brain. Release probability (P(r)) varies greatly between synapses. Understanding this fundamental feature of neurotransmission has implications for several basic and translational aspects of neuroscience: P(r) serves as gating node for information transfer between neurons and an understanding of P(r) is required to understand information flow; P(r) is implicated in maintaining healthy network activity; understanding dynamic regulation of P(r) will enhance understanding of plasticity within the healthy brain. This aligns with the mission of NINDS by contributing to an understanding of healthy physiology which may become perturbed in neurological and neuropsychiatric disorders. I will use novel imaging methods to investigate spatiotemporal P(r) regulation and how it is altered by arousal and neuromodulatory systems. P(r) is a core determinant of how information flow from pre-synaptic to post-synaptic cells (Fernandez- Chacon et al. 2001, Kaeser et al. 2013, Korber et al. 2016). In vitro and ex vivo experiments have revealed a wealth of information on how P(r) is dynamically regulated by various neuromodulatory and circuit functions (Higley et al. 2009, Munoz et al. 2014) and how it differs for synapses formed by the same axon (Reyes et al. 1998, Markram et al. 1998, Koester et al. 2005, Martinetti et al. 2022). The overall goals addressed in aims 1 of this proposal are to extend these observations to the awake, behaving brain by leveraging novel tools. In addition to functions localized at the synapse, global behavioral state as measured by pupil diameter, whisking, and locomotion has been extensively studied in relation to cortical dynamics and neuromodulation via acetylcholine (ACh) (Vinck et al. 2015; Lohani et. al. 2021; Benisty et al. 2021; Tang and Higley 2020; McCormick et al. 2020). Further, ACh interacts directly with muscarinic receptors on pre-synaptic terminals to influence glutamate release in part via Gi/o coupled signaling (Maeda et al. 2020; Higley et al. 2009; Guo et al. 2012). Leveraging the ability to quantify P(r) in vivo, I will probe interactions between state, ACh, and P(r) at cortical synapses during behavior. Aim 2 will interrogate behavioral state and ACh mediated neuromodulation. The laboratory of Dr. Higley is an excellent environment in which to conduct this investigation. Dr. Higley has extensive experience with synaptic release and plasticity as well as in vivo imaging methods. In addition, the Neuroscience Department at Yale boasts a highly-collaborative set of leading experts on synaptic physiology and biochemistry who will serve as advisors and mentors concurrent with the goals of the Training Plan herein.

NIH Research Projects · FY 2026 · 2023-12

Nitrogen fixation by the metalloenzyme nitrogenase supports nearly 50% of the global population and is the only biological pathway for nitrogen reduction. Nitrogenase consists of two proteins, the obligate reductase Fe-protein and the catalytic Mo Fe-protein, both of which are rapidly inactivated by oxygen. Biochemical, crystallographic, and spectroscopic studies have yielded seminal insights into the structures and functions of purified nitrogenase proteins, but a unified understanding of the enzymatic mechanism is still unrealized. These proteins are expressed in a small subset of prokaryotes termed diazotrophs, including aerobes and anaerobes, which inhabit a diverse array of environments. Despite this evolutionary demonstration of compatibility between nitrogen fixation and a variety of metabolisms, robust heterologous expression of the nitrogenase proteins has not been achieved. Instead, endogenous expression within the free-living soil bacterium Azotobacter vine land ii remains the most widely used system for the purification and study of nitrogenase. Several peculiar features of this obligate aerobe have been annotated under nitrogen-limited conditions, to wit, the formation of an intracytoplasmic membrane network, but a comprehensive investigation of these features and their relationship to the nitrogenase proteins is lacking. The primary hypothesis of this proposal is that interactions with cellular ultrastructures and as yet unidentified binding partners in vivo significantly regulate and promote nitrogenase activity. My goal is to identify and characterize these states of nitrogenase in diazotrophic organisms by training in and applying emerging cryoelectron tomography (cryoET) methodologies and performing experiments outlined in two Aims. Aim 1: Revealing the architecture of the nitrogenase interactome in A vinelandii with mass spectrometry and single particle (SP) cryoEM. Aim 2: Determine in situ structures of nitrogenase complexes in conjunction with cellular features using cryoET and sub-tomogram averaging (STA). As a postdoctoral fellow I gained expertise in anaerobic SP cryoEM to obtain high resolution structures of nitrogenase. These skills will provide a foundation for the proposed research. As I begin my independent career, I seek to leverage and apply training in the growing field of cryoET for the study of in situ structures. Throughout the outlined aims, I will also apply my training in genetic manipulation of non-model organisms, mass spectrometry, and cryoET, relying on local CryoEM and proteomic resources, and on national facilities, while pursuing new discoveries in SP cryoEM of

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Aedes albopictus (Ae. albopictus), also known as the Asian tiger mosquito, is a highly invasive and aggressive disease vector that has rapidly invaded every continent on earth except Antarctica. Ae. albopictus spreads diseases including Zika, Chikungunya, yellow fever, and dengue. Despite its immediate threat to human health, Ae. albopictus is highly understudied. Studies of other mosquitoes have focused on investigating how they integrate olfactory, visual, and thermal cues to guide their attraction to a blood-meal host. By contrast, there has been remarkably little study of mosquito taste systems, even though they may play a role in gating the final behavioral decision to bite a host. In particular, virtually nothing is known about taste in Ae. albopictus, a disease vector now in the US whose range is expanding rapidly because it outcompetes other species and because of climate change. Here we propose to use a systematic and multidisciplinary approach to test the hypothesis that Ae. albopictus uses its taste system to detect taste cues and guide biting behaviors. Because one of the most effective ways to prevent the spread of vector-borne pathogens is through prevention of biting, more in-depth investigation of taste cues influencing biting behaviors may aid in development of improved vector control methods. Using novel tools, we will examine new aspects of chemical cues, neuronal taste coding, and receptors that affect biting behaviors. To investigate the cellular basis of taste detection, we will systematically test the physiological responses of taste neurons to a panel of taste compounds, including blends of host cues with varying compositions. We will examine the effect of host cues on biting behaviors. This may shed light on the age-old question of why some people get bitten more than others by mosquitoes. We will functionally test the genetic basis of host detection by generating transgenic CRISPR-Cas9 mutant mosquitoes. In summary, we aim to elucidate the mechanism of an understudied mosquito sensory system in a species that is highly invasive and dangerous yet has received relatively little attention. The mentoring team and Yale University provide ideal support for Dr. Baik’s path to independence. The research and career development objectives outlined in this proposal will provide the training needed for Dr. Baik to secure an Assistant Professor position by the end of the K99 phase, and to apply for R01 funding as an independent investigator.

NIH Research Projects · FY 2024 · 2023-11

Project Summary Data sharing is essential to modern biomedical data science. Access to a large amount of genomic and clinical data can help us better understand human genetics and its impact on health and disease. However, the sensitive nature of biomedical information presents a key bottleneck in data sharing and collection efforts, limiting the utility of these data for science. The goal of this project is to leverage cutting-edge advances in cryptography and information theory to develop innovative computational frameworks for privacy-preserving sharing and analysis of biomedical data. We will draw upon our recent success in developing secure pipelines for collaborative biomedical analyses to address the imminent need to share sensitive data securely and at scale. Practical adoption of existing privacy-preserving techniques in biomedicine has thus far been largely limited due to two major pitfalls, which this project overcomes with novel technical advances. First, emerging cryptographic data sharing frameworks, which promise to enable collaborative analysis pipelines that securely combine data across multiple institutions with theoretical privacy guarantees, are too costly to support complex and large-scale computations required in biomedical analyses. In this project, we will build upon recent advances in cryptography (e.g., secure distributed computation, pseudorandom correlation, zero-knowledge proofs) to significantly enhance the scalability and security of cryptographic biomedical data sharing pipelines. Second, existing approaches that locally transform data to protect sensitive information before sharing (e.g. de-identification techniques) either offer insufficient levels of protection or require excessive perturbation in order to ensure privacy. We will draw upon recent tools from information theory to develop effective local privacy protection methods that achieve superior utility-privacy tradeoffs on a range of biomedical data including genomes, transcriptomes, and medical images by directly exploiting the latent correlation structure of the data. To promote the use of our privacy techniques, we will create production-grade software of our tools and publicly release them. We will also actively participate in international standard-setting organizations in genomics, e.g. GA4GH and ICDA, to incorporate our insights into community guidelines for biomedical privacy. Successful completion of these aims will result in computational methods and software tools that open the door to secure sharing and analysis of massive sets of sensitive genomic and clinical data. Our long-term goal is to broadly enable data sharing and collaboration efforts in biomedicine, thus empowering researchers to better understand the molecular basis of human health and to drive translation of new biological insights to the clinic.

NIH Research Projects · FY 2026 · 2023-11

PROJECT SUMMARY In mammalian cells, RNAs are predominantly located within the cytoplasm and nucleus. A recent study, however, has found that RNAs can be glycosylated, and a fraction of such glycosylated RNAs (referred to as glycoRNAs) are located on the outer cell surface. As outer cell surface represents a different topological space compared to cytoplasm and nucleus, these exciting findings raise important questions on the functions of cell surface RNAs, their precise chemical nature, their mechanisms of action, and mechanisms of their presence on the cell surface, all of which are not known. This proposal focuses on neutrophils, an important hematopoietic cell type that play important innate immune functions and are often the first responders toward inflammation and tissue damage. While the circulation half-lives of neutrophils are relatively short, neutrophils can migrate from circulation into tissues, a process that involves complex interactions with the endothelial cell layer lining the blood vessels. Glycan-binding lectins and integrins are known players in neutrophil-endothelium interactions, but the whole process remains incompletely understood. Based on our preliminary data, we propose the existence of glycoRNAs on neutrophil cell surface and aim to test that neutrophil cell surface RNAs mediate important functions of neutrophils including their interaction with endothelial cells. With a team of investigators of complimentary expertise, we will achieve these goals through four specific aims to decipher the molecular nature, functions, and mechanisms of neutrophil cell surface RNAs. Successful execution of our proposed project will add a new dimension to the regulation of neutrophil functions by studying a new class of RNA-containing regulators. The proposed experiments will also address key questions regarding glycoRNAs. As the field of cell surface RNA/glycoRNA is at its infancy, there are many exciting questions that cannot be explored in this single proposal, but findings from this proposal could stimulate both future explorations and the community toward better understanding of glycoRNAs in neutrophils and in other diverse biological settings.

NIH Research Projects · FY 2025 · 2023-11

PROJECT SUMMARY Pathogenic copy number variants (pCNVs) are strongly associated with neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorders and schizophrenia, yet identifying the genes or combinations of genes within pCNVs that drive syndromic phenotypes remains a central challenge. Cumulative data from our team and others has shown that the 3q29 deletion is associated with a high neurodevelopmental and neuropsychiatric illness burden, including a 40-fold increase in schizophrenia risk. We identified reduced cerebellar volume as a core phenotype that correlates with greater phenotypic severity including lower IQ. However, the individual genes driving these phenotypes remain unknown. In this proposal, we will use a highly innovative approach that capitalizes on the unique features of zebrafish to rapidly screen the function of genes and combinations of genes in the 3q29 deletion in the developing vertebrate brain. Our central goal is to identify the driver genes and/or gene combinations that are associated with neurodevelopmental phenotypes. Our central hypothesis is that individual driver genes in the 3q29 deletion interval act alone or in smaller subsets to cause reduction in cerebellar volume, growth deficits, and behavioral dysfunction. This hypothesis is based on accumulating evidence supporting the presence of driver genes for specific phenotypes in pCNVs, as well as the high expression level of multiple genes in the 3q29 interval in the cerebellum. To test this hypothesis, we will perform in vivo screens of individual 3q29 genes in zebrafish using a novel CRISPR-F0 method that allows us to rapidly assess the effects of gene loss of function. We will assess the consequences of loss of function of individual genes on body size, brain structure, and cerebellar volume using a custom whole-brain mapping pipeline (Aim 1) and basic arousal and sensory processing behaviors using automated, high-throughput assays (Aim 2), and assess the effect of disrupting combinations of genes on these phenotypes (Aim 3). The expected outcome of this research is to identify driver genes and/or gene combinations in the 3q29 deletion that contribute to a range of neurodevelopmental phenotypes. The broader impact of this research is that it will provide a path forward for the identification of driver genes and gene interactions that is generalizable across pCNVs associated with neurodevelopmental and neuropsychiatric disorders.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Opioid use disorder (OUD) is a significant public health problem in the United States, with overdoses and deaths currently maintaining at epidemic levels. As risk of overdose is highest following relapse and treatment dropout, improved mechanistic understanding of risk and protective factors in individuals currently receiving medications for OUD (MOUD) is urgently needed. To address this, this Cutting-Edge, Basic Science Award (CEBRA) application moves beyond the limitations of traditional case-control designs to rapidly advance our understanding of the neural mechanisms of early MOUD treatment. For decades, clinical neuroimaging has relied on case-control designs in which individuals with a given psychiatric disorder are compared to a group of matched healthy ‘control’ individuals, or a group of individuals distinguished by another individual difference feature. While informative, these approaches by definition focus on group average deviations from a presumed normative population and thus may have relatively limited real world clinical utility. For example, recent findings from machine learning studies of addictions and other disorders indicate that brain networks which distinguish patients from controls are often distinct from brain networks that predict specific clinical outcomes within-group. This striking distinction suggests that person-specific neurobiology is dissociable from group-specific patterns, and thus group-specific findings are unlikely to translate to an improved understanding of person-specific pathophysiology or to treatment. Our innovation, the characterization of neural trajectories during MOUD treatment using dense sampling (i.e., repeated longitudinal assessments of the same individual) will provide unprecedented mechanistic insight into the neurobiological basis of OUD remission. Dense sampling is an emerging methodology that aims to overcome limitations with cross-sectional research that inherently assumes the brain is static and unchanging. This approach is particularly relevant to studying MOUD treatment: MOUD is multiphasic, comprised of medication induction, stabilization, ongoing treatment and eventual discontinuation phases. However, with a few small exceptions, existing neuroimaging efforts are almost exclusively single time-point assessments which, by definition, fail to capture dynamic trajectories of individual risk and resilience that can be used to mechanistically inform treatment advancements. This pilot project therefore applies dense sampling to characterize early trajectories of MOUD recovery at unprecedented temporal resolution—i.e., bi-weekly over three months. To maximize mechanistic insight, complementary clinical and behavioral computational data will also be acquired longitudinally. Borrowing from basic human neuroscience, our dense sampling neuroimaging approach represents a paradigm shift for psychiatry research in general, and holds enormous potential to inform understanding of opioid use disorder remission specifically.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Diagnostic errors, delays, and related problems have been shown to be widespread in outpatient settings. Patient and family reports of these experiences offer a vital perspective on the scope, causes, and consequences of these mishaps. Yet, to date, information regarding patient experiences has been quite limited in the U.S., largely involving inpatient treatment or limited samples of patients from a few health systems or self-selected patient advocacy groups. This project would provide the first nationally representative estimates of Americans’ experiences with and concerns about diagnostic safety in ambulatory care. Deploying a survey incorporating several innovative survey methods, the study would (a) more reliably identify patient-reported safety events than in past surveys, (b) more fully elicit patient- and family-reported narrative accounts of those events, and (c) more consistently track the extended consequences of diagnostic problems. Data would be collected from 3,300 U.S. households that experienced diagnostic problems between 2020 and 2025. A sample this size makes possible comparisons across multiple settings (including newly emergent sites like urgent care clinics) and among different subgroups of patients, including those most marginalized historically. Because establishing an accurate, timely diagnosis is often challenging for clinicians and confounding to patients and their families, diagnostic problems are common in every healthcare system. But American healthcare financing and delivery systems are especially fragmented. We hypothesize that this fragmentation elevates the risks and harms of diagnostic breakdowns, particularly in outpatient settings. The study will test these hypothesis for three forms of insecurity induced by fragmentation (a) coverage insecurity, linked to anxieties about what healthcare insurance will cover, (b) guidance insecurity, emerging from patients’ difficulty identifying a clinician who can help them navigate the diagnostic process, and (c) process insecurity, emerging when multiple clinicians and care settings are associated with the diagnosis, leaving patients with inconsistent expectations for how the diagnostic process will unfold. Because all three forms of insecurity vary greatly within the American healthcare, we can both empirically assess the impact of fragmentation on diagnostic outcomes and estimate the potential benefits of reducing these three forms of fragmentation.

NIH Research Projects · FY 2026 · 2023-09

Abstract The goal of this proposal is to understand how visual signals are transmitted from the outer to the inner retina through cone bipolar cells (CBC), with a focus on detailed synaptic mechanisms and functional circuits between morphologically and physiologically identified cone bipolar cell types and specific ganglion and amacrine cell types in the whole-mount mouse retina. This investigation is motivated by an important observation that, although recent transcriptomic, connectomic, and imaging studies have significantly advanced our understanding of bipolar cell classification and anatomical structure, our knowledge of the detailed physiology of the synapses and circuits formed by these classified CBC types remains very limited. To address this fundamental gap in our understanding of retinal processing, we developed a new experimental approach, using dual pre- and post-synaptic patch-clamp recording from pairs of morphologically identified CBC types and ganglion cell types in the whole-mount mouse retina in conjunction with two-photon optical recording and targeted expression of genetically encoded glutamate and Ca sensors. This approach allowed us to correlate the anatomical structure of each morphological CBC type with its intrinsic and receptive-field physiology and, more importantly, to directly measure, at a millisecond resolution and under voltage-clamp condition, synaptic transmission from identified CBC types to their postsynaptic targets in a structurally intact retina. Our preliminary results revealed novel kinetic and circuit properties of CBC that suggested a new mechanism of synaptic integration using both chemical and electrical synaptic transmission. Based on these preliminary results, we propose a dual-mode synaptic mechanism by which CBCs transmit both a direct synaptic signal and a gap junction-coupled network signal to their postsynaptic targets. This hypothesized mechanism will be tested through the three Specific Aims. Aim 1, dual patch-clamp characterization of synaptic transmission and functional connectivity between morphologically identified CBC types and a diffused ganglion cell type (W3) in the whole-mount retina. Aim 2, understand the chemical and electrical synaptic interactions underlying signal transmission from identified CBC types to W3 cells in the whole-mount retina. Aim 3, determine the functional impact of different modes of CBC synaptic transmission on diffused and narrowly stratified postsynaptic target cells. Results from this study are expected to provide novel insights into the synaptic mechanisms and functional circuitry of cone bipolar cell types in the mammalian retina and shed light on chemical and electrical synaptic integration in the CNS in general.

NIH Research Projects · FY 2026 · 2023-09

Genetics of Cannabis Use Disorder and Cannabinoid Response In Humans Cannabis is widely used worldwide and is associated with negative outcomes including cannabis use disorder (CanUD), psychosis, and cognitive impairment amongst others. Given the legalization of “recreational” and “medical” cannabis globally, the increasing availability of cannabis, the higher potency of cannabis, the availability of highly potent cannabinoid products, the commercialization of cannabis, and the rising rates of cannabis use, it is critical to understand how genetic factors influence 1) an individual's vulnerability for addiction and psychosis, 2) the response to cannabinoids, 3) the response to novel treatments for CanUD.CUD is strongly genetically influenced; we published the first CUD genomewide association study (GWAS) with genomewide-significant results; however, the precise nature of the contribution of genetic factors in the development of CUD is still not clear. Cannabis exposure has also been linked to a number of psychosis outcomes including schizophrenia (SCZ). SCZ is highly heritable and population-based and genetics studies both support a bidirectional genetic relationship between SCZ and CanUD. However, the precise contribution of genetic factors in the development of psychosis outcomes related to cannabis are not clear. We propose a translational research program bringing together two highly productive and complementary research groups (genetics [Gelernter] and cannabinoid pharmacology [D'Souza]). 1) We will conduct a genomewide association analyses and meta-analyses of CanUD to compute PRS for CanUD (CanUD-PRS) based on best- and largest-available datasets. that includes existing and new data releases of MVP, AllofUs; FinnGen and other relevant data that becomes available over the course of the project. We will study genetic overlap and causality of CanUD with other complex traits in EA, AA, and other ancestry groups. 2) We will also determine the extent to which CanUD-PRS and SCZ-PRS influence the acute effects of Δ9-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis in a Human Lab Study (HLS). Lastlywe will explore the extent to which 3a) CanUD-PRS predicts response to FAAH-Inhibitor treatment and also severity of CanUD pretreatment in a completed NIDA funded trial, and 3b) CanUD-PRS and SCZ-PRS, respectively influence the efficacy and safety of cannabis derivatives in a prospective large VA-funded multicenter trial with cannabinoids. Successful completion of this study is expected to 1) identify many more genetic risk loci for CUD, 2) in European and non-European populations, 3) with post-GWAS statistical analysis to understand the biologyof CUD, 4)translation via human experimental studies to increase our understanding of the response to THC infusion, and its implications for the development of cannabis use disorder and psychosis, and 5) the genetic influence on response to novel treatments for cannabis use disorder and cannabinoid treatments for neuropathic pain.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Active perception, the ability to seek out behaviorally relevant information, is guided by both cognitive and motor behaviors and is influenced by fluctuations in endogenous brain state. It is a result of the concerted activity of ensembles of neurons in the sensory hierarchy. These ensembles interact flexibly and dynamically as the organism transitions between various behavioral and brain states. However, the state-dependent information processing principles that underlie the activity of such ensembles are largely unknown. A rich body of theoretical and recent experimental work has shown that dependencies, such as co-variability, within an ensemble strongly influence their information carrying capacity and hence their functional efficacy. Further, theoretical investigation of these dependencies has revealed that their influence is determined by the extent of their alignment with the information coding dimension of an ensemble. The source of dependencies – shared vs. local – has been identified as a key determinant of this alignment. A critical step toward determining this source is to characterize the joint spiking activity (beyond pairwise correlations) of populations in these ensembles. This is, however, a challenging task owing to the varied non-linear nature of neuronal interactions, and the fact that neural populations are sparsely sampled by current recording techniques. Tackling this problem requires a highly interdisciplinary approach spanning advanced techniques in systems and computational neuroscience. Based on our preliminary data and prior studies, our broad hypothesis is that the computations of active perception are cortical layer-specific and that they are mediated by ensembles of neural sub-populations defined by their layer identity and cell-class. We propose to answer several key questions regarding this hypothesis: how does active perception modulate information flow in laminar circuits, both during attention (Aim 1A) and saccadic eye movement (Aim 1B)? How are the laminar circuits of active perception modulated by internal brain state fluctuations such as those during cued attention (Aim 2A) and spontaneous vision (Aim 2B)? We will achieve these aims using laminar high-density recordings in the visual cortex of non-human primates, while animals are engaged in either task-based or spontaneous active perception. Using a novel combination of dynamic Bayesian networks among (DBN) and partial information decomposition (PID) we will infer distinct categories of dependencies cortical network components. Our proposal will achieve the first systematic characterization of modulation of information flow (beyond pairwise correlations) by active perception processes in the context of laminar cortical circuits. Our proposal is the first study to investigate how internal brain state fluctuations shape the ensemble level causal motifs of active perception. The results of these investigations will significantly advance our understanding of information flow structure in a canonical cortical circuit and provide a broad framework for investigating such structures in brain-wide circuits.

NIH Research Projects · FY 2025 · 2023-09

Women with opioid use disorder (OUD) are disproportionately impacted by intimate partner violence (IPV) and PTSD, with up to 78% of women receiving medication for opioid use disorder (MOUD) treatment experiencing IPV in the past 6 months. Regardless of PTSD diagnosis, 72%-78% of W-IPV experience clinically significant PTSD-related impairment in functioning. PTSD reduces treatment retention. Interventions effectively target PTSD to reduce substance, but many are not advised for IPV because they are exposure-based therapies for trauma that occurred in the past – not for trauma that is ongoing, as with IPV. Integrated interventions for other health conditions improve outcomes for MOUD treatment, yet no evidence-based treatments exist that integrate a much-needed focus on IPV and PTSD into MOUD treatment. Present-Centered Therapy+ (PCT+) and Helping to Overcome PTSD through Empowerment (HOPE) are two evidence-based, manualized behavioral interventions designed for women experiencing IPV (W-IPV) to reduce PTSD symptoms and other trauma-related outcomes. PCT+ focuses on helping W-IPV cope with current stressors that arise from their traumatic experiences; it can be delivered as an 8 session treatment by nonclinical, professional staff who may be more available and affordable in MOUD treatment settings. Some women may not respond to PCT+ alone and need additional treatment. HOPE is an IPV-specific cognitive behavioral therapy delivered in 10 sessions by Master’s level therapists and incorporates empowerment and stabilization treatment models. Our innovative approach packages these two interventions in a stepped care model to create PCT+2HOPE. This phased study conducted in three MOUD treatment settings in the United States northeast will be guided by the Exploration, Preparation, Implementation, and Sustainment (EPIS) framework. We will explore, prepare for, and then implement a randomized controlled trial to evaluate PCT+2HOPE versus treatment as usual while collecting data relevant for informing future sustainment. We will elicit input from W-IPV as well as direct service and supervisory staff to inform: protocols for systematically identifying IPV in OUD treatment settings, processes for referral to domestic violence service providers, and adaptation of the PCT+ and HOPE interventions (R61 phase). Then, we will evaluate the impact of PCT+2HOPE on promoting retention in MOUD treatment while improving secondary outcomes (e.g, PTSD-related impairment in functioning and client-defined recovery) (R33 phase). We will explore whether the effectiveness of the interventions differ based on access to basic needs. Building on established partnerships, our interdisciplinary study team includes community-based OUD and domestic violence service providers, and academic partners with expertise in IPV, PTSD and OUD-related care; addiction medicine; clinical trials with adaptive designs; community-partnered research; and implementation science. Our study has potential for high impact by generating data on a reproducible and scalable approach that may transform treatment for the unique needs of W-IPV with OUD.

NIH Research Projects · FY 2025 · 2023-09

The opioid crisis continues to evolve, creating ongoing challenges for timely and effective care delivery. To support more consistent treatment practices, implementation research must adapt quickly to emerging evidence. Clinical decision support (CDS) offers a scalable strategy to accelerate the adoption of evidence-based practices across healthcare systems. Patients often become more receptive to treatment following key clinical encounters, such as an emergency department (ED) visit for opioid overdose. However, treatment initiation in these settings remains inconsistent. To address this gap, we conducted the EMBED pragmatic, cluster-randomized trial. This study evaluated a non-interruptive, electronic health record (EHR)-based CDS to support patient assessment and automate EHR workflows related to initiating buprenorphine during routine ED care for individuals diagnosed with opioid use disorder (OUD). The CDS increased the proportion of physicians who initiated buprenorphine, resulting in its national dissemination. Post-trial analyses revealed variation in treatment patterns and highlighted opportunities to expand reach and increase adoption. However, most CDS tools remain static during evaluation, and traditional assessment methods are resource-intensive and slow, limiting the ability to rapidly improve and implement effective interventions. These challenges must be addressed to accelerate the delivery of data-driven solutions for the opioid crisis, including the continued optimization of the EMBED CDS. The EHR environment not only enables CDS delivery but also provides a scalable, low-burden method for evaluating care processes using automated log data. Current CDS performance metrics, such as alert dismissal rates, are insufficient for assessing interface design, workflow integration, and real-time uptake. To address this gap, we will apply a Multiphase Optimization Strategy (MOST) framework using rapid, iterative randomized testing and pragmatic EHR-based metrics to pursue the following aims: (1) Refine and validate reproducible, scalable outcome measures for CDS uptake and usability related to ED-initiated buprenorphine for OUD, and (2) Refine and test a multicomponent CDS intervention to improve ED-initiation of buprenorphine through enhanced CDS adoption, interface usability, and system reach. Achieving these aims will establish a path toward scalable, adaptive interventions for improving treatment delivery in the context of the opioid crisis. Dr. Melnick and the ADAPT team bring extensive experience in emergency medicine, clinical decision support, implementation research, biostatistics, and health systems evaluation, positioning them to execute this work successfully.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT This application seeks three years of dissertation funding to have a computational PhD candidate confront a pressing public health issue using three interrelated, interdisciplinary aims supported by concomitant mentorship and training that will prepare them for a future academic career in using statistical, computational and mixed- methods techniques to develop and analyze interventions to address the opioid crisis. Over 9.5 million people reported using opioids in the US in 2020, during which time there were ~257 opioid-related overdose deaths per day. Fatal overdose rates have grown nationally by 274% from 2013 to 2020, largely due to the growing presence of synthetic opioids and other additives, primarily found in street drug supplies. Street-obtained substances with unexpected composition, such as cocaine containing fentanyl or heroin with novel fentanyl and benzodiazepine derivatives, have been causing overdoses en masse. To mitigate the scale of these mass injury events, over 3000 agencies in 49 states use the Overdose Detection Mapping Application Program [ODMAP], which features a “spike alert”-based warning system. ODMAP issues a spike alert when overdose counts exceed preset thresholds within 24 hours to help mobilize rapid public health responses to prevent overdoses and save lives. The state of Connecticut has one of the highest overdose rates in the country, with 39.1 out of every 100,000 people experiencing a fatal overdose in 2020. To address this crisis, it has implemented one of the most progressive evidence-based overdose spike response systems in the nation, with each ODMAP spike alert undergoing an extensive manual review by the Department of Public Health that occasionally culminates in a public health alert. The effectiveness of this system rests on its ability to accurately identify spikes and rapidly mobilize a public health response to save lives, but it is unclear 1) if the system has any effect on overdose rates 2) how first responders, harm reduction organizations and health systems make use of the system to rapidly respond to overdose spikes 3) if the system can be modified to more accurately identify spikes and motivate rapid responses to save lives. I therefore propose to 1) estimate the causal effect of Connecticut’s current spike alert system on subsequent overdose-related outcomes; 2) assess utilization of the current system, barriers to overdose prevention and opinions on alternatives to the status quo; and 3) develop and simulate the impact of alternative spike alert strategies on overdose-related outcomes. To address these Aims, I will use a combination of cutting-edge causal inference, mixed-methods, space-time regression and epidemiological modeling techniques, along with integrated data sources and guidance from key stakeholders. These findings will provide actionable advice to improve Connecticut’s current spike alert system, can motivate future policy work to address the overdose crisis and provide a framework for other health departments looking to implement spike alert systems that are responsive to stakeholder needs and can more effectively save lives than the status quo.