University Of Texas At Austin

NIH Research Projects · FY 2026 · 2023-01

Flow cytometry is the tool of choice for high-speed analysis of large cell populations, with the tradeoff of lacking intracellular spatial information. Imaging flow cytometry (IFC) has emerged as a new tool that combines advantages of microscopy with the high speed of flow cytometry. However, they can only provide 2D images to determine three-dimensional (3D) distribution of cellular features, have a limited field of view (FOV), and require precise control of the fluidic system to minimize image blurring due to uncontrolled cell rotation or translation across the FOV. The absence of 3D imaging results in ambiguity of object locations and blurring by focal depth due to the projection of a 3D cell into a 2D image. Although in the last decades flow cytometry systems that can actually acquire three-dimensional (3D) spatial information were developed, constraints related to resolution and samples size remained as their biggest limitation. Therefore, the goal of this proposal is to develop the next generation 3D imaging flow cytometers with high-throughput and high-content capabilities for 3D imaging of hundreds to thousands of cells and spheroids per second with high resolution, for the first time. We propose to develop such a cytometry method, using a novel microscopy method, Line Excitation Array Detection microscopy (LEAD), that can image objects in large field of views at the rate of current 1D cytometers, but with high 3D resolution and high signal-to-noise ratios (SNR). Our proposed LEAD cytometer is a fast-scanned light-sheet microscope capable of MHz frame rates. We will develop the fastest MHz line-scanning method using a longitudinal acousto-optic deflector driven by a chirped frequency signal. We will image the scanned light sheet using a linear silicon photomultiplier array, which will provide the sensitivity required when scanning so quickly, and the parallel readout required for such high frame rates. First, we will develop linear LEAD 3D imaging flow cytometry at sub-micron scale resolution and small FOVs. Although our preliminary data indicates we will be able to image at 100 kHz – MHz frame rates at such high resolution with high SNR, we will perform experiments measuring the SNR to determine the operating range of LEAD cytometry. In the second aim, we will increase the FOV by developing two-photon LEAD imaging flow cytometry with Bessel beams. To support the larger FOV, we will develop a 128-channel data acquisition system using eight 16-channel data acquisition cards. In the third aim, we will develop a state-of-the-art computational infrastructure that allows for file transfers up to 25 GB/s, storage (>100 TB), and analysis that only takes 3x the imaging time. We will use 2 deep learning models for analysis. If successful, this high-risk/high-reward proposal would alter the imaging flow cytometry landscape. The proposed 3D imaging flow cytometer can offer improved cell and spheroid analysis in diverse biomedical fields such as cancer biology, microbiology, immunology, hematology, and stem cell biology. Improved sensitivity will help users to improve research outcomes or diagnose patients with higher statistical power.

NIH Research Projects · FY 2026 · 2023-01

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that catalyze phosphorylation of diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA serve as potent lipid messengers to shape cellular responses by altering subcellular localization, activation, and function of essential receptor proteins (ranging from enzymes to transcription factors). DAG and PA also serve as building blocks for phospholipid and triglyceride biosynthesis and integral to membrane architecture and bioenergetics. The significance of our proposed studies is the enormous therapeutic potential of targeting individual DGKs because of their fundamental role in sculpting the lipidome to support metabolic, structural, and signaling demands of healthy and diseased cells. Despite their clinical value and discovery nearly 30 years ago, gaps in knowledge with regards to ligand binding and regulation of DGK active-sites in living systems have confounded basic understanding of how 10 mammalian DGK isoforms, which share a common catalytic domain, are capable of regulating distinct metabolic and signaling functions. We will test our hypothesis that C1 and other non-catalytic domains, which largely differentiate DGK isoforms, function in substrate and inhibitor recognition of DGK active sites. The proposed research program will test whether selective blockade of DGK can restore deficient DAG signaling to overcome immunosuppression of tumor infiltrating lymphocyte activity. Genetic and clinical evidence point to DGKs as promising targets for reversing immunosuppression of T cells although the molecular mechanisms coupling disrupted DGK metabolism to enhanced TCR signaling are not clear. Our mechanistic studies will establish a testable model for fundamental understanding of substrate and inhibitor recognition in DGK active sites to guide development of new chemical strategies to perturb activity of T cell specific DGKs in vivo for immunotherapy applications. Our long-term goals for this proposal are to functionally map novel and druggable small molecule binding sites on DGK and potentially other DGK isoforms in T cells to: 1) gain molecular level insights into DAG fatty acyl chain recognition and specificity, 2) identify molecular features of enzyme active sites to target lipid versus protein kinases, and 3) develop new inhibitors for selective inactivation of DGK isoforms in live cells and animals. We will test 2 independent yet related specific aims directed at: (Aim 1) identification of the DAG binding site, (Aim 1) understanding how individual DGK domains couple extracellular signals to shape T cell responses, (Aim 2) determining how DGK inhibitors amplify T cell activation, (Aim 2) understanding how DGK inhibitors reverse T cell immunosuppression in vivo, and (Aim 2) determining if DGK inhibitors affect membrane translocation. The overall impact of our findings will be to understand how intrinsic features of DGKs cross-talk with extrinsic features of cellular environments to form the basis of a lipid signaling code that can be therapeutically targeted for reversing immunosuppression of T cells.

NIH Research Projects · FY 2026 · 2023-01

Abstract The development of new protein biosensors has for the most part been dependent on finding a protein that is already responsive to a known effector. While rational design and directed evolution methods exist for altering the effector specificity of transcription factors, these methods are in general complex and slow, and have failed to solve the more general problem of identifying new protein biosensors at will. In particular, it is often difficult to find a receptor that is both sensitive and specific for a given end product or intermediate, and even when efforts to generate new sensors are successful, they generally recognize effectors that are structurally quite similar to their natural counterparts. In particular, for virtually all industrially and medically useful terpenes there exists no corresponding biosensor. We now propose to develop a combined computational and directed evolution method that should allow us to proceed from any of a wide variety of ‘generalist’ repressors to create highly sensitive and specific biosensors for a structurally diverse range of terpenes and terpenoids for which no biosensors are currently known. To this end, we have developed a novel directed evolution method for altering biosensor specificities, and propose to synergize these with powerful machine learning tools for improving protein function. Extensive Preliminary Results show that the TetR family of transcription factors can be readily manipulated to take on new effector specificities, and that machine learning can be used to improve the function of a wide variety of proteins. We now further propose to identify semi-specific transcription factors as starting points for biosensor design and evolution (Aim 1); use neural network approaches to predict new sensor specificities (Aim 2); and refine these predictions via directed evolution and high-throughput screening (Aim 3).

NIH Research Projects · FY 2026 · 2023-01

Injectable Hydrogel Electrodes to Prevent Ventricular Arrhythmias In the United States, sudden cardiac death accounts for 350,000 deaths per year with the leading cause being lethal ventricular arrhythmias. The underlying electrophysiologic derangement mechanistically responsible for ventricular arrhythmias is delayed conduction velocity in scarred or otherwise diseased myocardium. Access to the smaller vessels and tributaries that cross over scarred region of the heart could provide improved pacing; however, there are no pacing leads small enough to navigate these smaller tributaries. In this research, we propose a novel method to treat and manage ventriculararrhythmias – developmentof a newconductive material that can fill both large and small coronary vessels and convert these tributaries into flexible electrodes to restore capture across regions of scarring. Our collaborative team that combines clinical expertise (Razavi) and biomaterial science (Cosgriff-Hernandez) has demonstrated early feasibility of pacing myocardium with an in situ curing hydrogel in a pig model. We plan to build on this initial proof of concept to develop a combined material and delivery system that can interface with existing pacemaker technology to greatly expand their capability to treat ventricular arrhythmias. Upon successful completion of these aims, we will have utilized a battery of in vitro and in vivo tests to establish the safety and efficacy of this new injectable hydrogel electrode. Confirmation of increased activation area as compared to standard-of-care single point pacing will validate the efficacy of this innovative approach to eliminate the conduction delay in scarred myocardium that results in lethal ventricular arrhythmias. We will use a post-myocardial infarct model to demonstrate that hydrogel electrode pacing reduces the frequency of ventricular arrhythmias and defibrillation shocks. Painless stimulation of wide areas of the heart using planar wavefront propagation from these hydrogel electrodes provides a new cardiac resynchronization therapy that will alter the landscape of cardiac rhythm management.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY The mediodorsal thalamus (MD) and its reciprocal connection with medial prefrontal cortex (mPFC) control important aspects of executive functioning and social behavior. Dysfunction of this neural circuit can cause developmental brain disorders and neuropsychiatric conditions. Understanding prefrontal thalamocortical (MD→mPFC) circuit function has been hampered by a lack of understanding of MD projection neuron types and how they integrate and process synaptic information. The central hypothesis is that differences in intrinsic properties and connectivity between the two major populations of neurons that provide ascending input to the mPFC causes them to extract and transmit different information to the mPFC. Thus, these two populations have different roles in behavior. The overall goal is to expand understanding of the circuits within MD. The rationale is that understanding of neural processing by the thalamus and thalamic inputs to prefrontal cortex is necessary for understanding the mechanisms of executive function and developing neuromodulation therapies targeting the prefrontal network for neuropsychiatric disorders. The central hypothesis will be tested with three specific aims: 1) Determine how intrinsic properties of MD neurons control synaptic integration of mPFC inputs. 2) Test how MD neurons differentially process synaptic inputs arising from different brain regions. 3) Determine how optogenetic manipulation of specific MD circuits affects cognitive, social, and affective behaviors in wildtype mice. The research proposed in this application is innovative, in the applicant's opinion, because it defines the function of an understudied but essential thalamic nucleus, from the level of membrane biophysics, to synaptic integration, to control of behavior. The work is significant because it will contribute to the anatomical and physiological map of prefrontal thalamocortical circuitry. Ultimately, such knowledge has the potential to guide the development of future neuromodulation strategies to treat the symptoms of neuropsychiatric disorders that localize to the prefrontal network.

NIH Research Projects · FY 2026 · 2022-12

Project Summary/Abstract Society at large is facing a global “dementia epidemic” that is predicted to intensify with the growing aging population. Although there is currently no cure for this devastating and pervasive condition, one life experience shown to protect against the onset of Alzheimer’s disease and related disorders (ADRD) is that of bilingualism. Nevertheless, findings regarding bilingualism as a contributor to cognitive reserve are mixed. These mixed findings may, in part, be attributed to variability in the characterization of the bilingual experience across distinct sociocultural contexts in addition to the presence of potential confounding variables. Little attention has been directed towards examining bilingualism factors (e.g., language dominance, proficiency, use and age of acquisition) that may underly these findings. In addition, limited attention has been directed towards language- prominent dementia syndromes, such as primary progressive aphasia (PPA). Although speech and language impairments are ubiquitous features of ADRD, in the case PPA these debilitating deficits manifest as initial, predominant symptoms. Differential patterns of language decline have been observed in bilingual PPA, with resilience of the first learned language and parallel decline reported in the literature. However, a nuanced account establishing patterns of language decline across different linguistic domains has yet to be systematically examined in PPA. In addition, although positive effects of speech-language intervention are now well documented in monolingual speakers with PPA, there is a significant gap in the literature examining treatment optimized for bilingual speakers with PPA. Moreover, the behavioral, neural and bilingual factors associated with language re-learning have yet to be established in bilingual speakers with PPA. The overall aim of the proposed research is to establish associations between bilingualism factors and the onset, decline, and treatment response of Hispanic, bilinguals (Spanish-Catalan) with PPA. In Aim 1, we seek to identify bilingualism factors associated with a later age of onset in Hispanics with each PPA variant. In Aim 2, we seek to identify the bilingualism factors associated with differential patterns of language impairment in Hispanics with PPA using metrics derived from connected speech. In Aim 3, we will evaluate the benefits of tailored speech-language intervention in the largest behavioral rehabilitation study of bilingual Hispanics with PPA to-date. We will also identify the bilingualism factors, pre-treatment cognitive-linguistic measures, and brain regions implicated in bilingualism that predict the magnitude of within-language gains and cross-language transfer effects. In order to accomplish these aims, we will enroll 90 Hispanic, bilinguals with PPA who will undergo behavioral assessment, MRI, and speech-language treatment. This proposal will provide needed evidence regarding cognitive reserve and linguistic resilience by leveraging a large bilingual PPA cohort via an established international collaboration. Outcomes will also provide crucial knowledge regarding neural mechanisms of language re-learning and will address how specific bilingual factors influence cognitive reserve and linguistic resilience in language-prominent dementia.

NIH Research Projects · FY 2025 · 2022-09

Education is among the most important determinants of later-life cognitive functioning and biological markers of AD/ADRD risk. However, we know very little about how, why, or for whom education matters for these cognitive outcomes. This makes it difficult to design effective early life preventative interventions. To know how and why education matters for later-life cognitive functioning and biological markers of AD/ADRD risk, we need high quality prospective studies that follow young people through schools and across adulthood; that measure key and modifiable educational contexts, opportunities, and outcomes; that observe midlife socioeconomic attainments; and that assess cognitive functioning and biological markers of AD/ADRD risk in late life. To date, no such studies exist. This project brings together an established and interdisciplinary team of neurologists, neuropsychologists, sociologists, education scientists, survey methodologists, biostatisticians, and neuroimaging experts who will re-contact surviving members the National Longitudinal Study of the High School Class of 1972 (NLS-72; N=14,489). NLS-72 is a nationally representative random sample of Americans first interviewed as high school seniors in 1972. Following protocols developed and successfully deployed in the High School and Beyond (HSB) cohort in 2014-2015 and 2021, the team will conduct in-home interviews that include extensive cognitive assessments and anthropometric measures; in-home visits to gather whole blood; and (for 500 people near one of five regional centers) neuroimaging via harmonized magnetic resonance imaging (MRI). The resulting data—which will be securely released to the wider scientific community during the project period—will be used to conduct transformative analyses of the effects of educational contexts, opportunities, and outcomes on risk of AD/ADRD as observed in cognitive assessments, blood-based markers of neuropathology, and neuroimaging. The project has four aims: (Aim 1) To estimate the extent to which education shapes biological, neurocognitive, and behavioral markers of AD/ADRD risk at age 70; (Aim 2) To estimate the extent to which adult socioeconomic attainments mediate the effects of education on vascular health, pace of biological aging, and cognitive functioning at age 70; (Aim 3) To estimate the extent to which racial and ethnic differences in the quality of and returns to education account for disparities in markers of AD/ADRD risk; and (Aim 4) To securely disseminate newly collected NLS-72 data for wide use by the research community. The analyses made possible by the newly collected data will transform our understanding of how and why education and other early life factors impact AD/ADRD risk and resilience.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Activation of ion channels upon binding multiple ligands at distinct subunits or domains is essential for synaptic transmission and cellular signaling. Despite recent advances in understanding their 3-dimensional structure, there remains for many channels a fundamental gap in our understanding of the sequence of events by which multiple binding sites and domains coordinate to open and close the channel pore. A major barrier to bridging this gap is that ensemble-averaged binding measures from many channels at once occlude observation of the distinct asynchronous binding steps that underlie the sequence of binding events at each individual channel. To overcome this barrier, I will use innovative single-molecule fluorescence methods developed in my lab in combination with my prior expertise with zero-mode waveguide nanophotonic arrays that enable optical tracking of each individual binding step. The objective of this proposal is to determine the energy landscape for 1) the sequence of stepwise binding events that drive activation of cyclic nucleotide gated (CNG) channels critical for visual and olfactory sensation, and 2) modulation of GABAA receptors by benzodiazepines (BZDs), one of the most widely prescribed psychotropic drugs today. The rationale is that optical tracking of individual binding events that are the chemical stimuli by which these channels operate will enable determination of the sequence of distinct energetic events that must at least partially occur prior to pore opening and thus are difficult to measure with electrophysiological approaches. The specific aims will: 1) Establish the energy landscape for sequential binding at a CNG channel; 2) Quantify the likelihood of CNG channel opening with each distinct binding step, which will test existing disparate model predictions; 3) Develop a mechanistic model for CNG channel activation that accounts for each distinct binding step; 4) Determine the energy landscape for BZD-binding or sequential agonist binding at GABAA receptors, and 5) Establish whether or not BZDs alter distinct agonist binding steps. The proposed research is significant because it will provide a necessary foundation for understanding the dynamic sequence of events governing ligand-driven behavior in these channels, which currently remain only poorly understood. The results will have an immediate positive impact as a quantitative benchmark for computational, structural, and functional studies aimed at uncovering the physical basis for the observed changes in energy. Ultimately, understanding the full sequence of events during channel activation is essential not only to advance our fundamental knowledge of ion channel mechanisms, but also to facilitate development of therapies targeting distinct steps in the activation pathway. Long-term, this knowledge will enable the rational design of new therapies to improve treatment outcomes and quality of life.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Biomolecular condensates are micrometer-scale membraneless structures that self-assemble in living cells. They are ubiquitous across diverse organisms and exist in protein-only conglomerates or protein associated with nucleic acids. Biomolecular condensate formation is critical to normal cellular processes and the mis-regulation of condensate assembly or disassembly drives various pathologies. We recently discovered that the pathological fusion protein EML4-ALK spontaneously forms micrometer-scale condensates in the cytoplasm while lacking conventional condensate-forming domains or sequence motifs. This condensate elicits a novel mode of cell signaling by acting as a physical platform that enriches signaling proteins. The recruitment of client proteins to elevate local protein concentrations implies a general strategy through which multi-protein condensates achieve biological function. A major gap in the field is that very little is known about condensate-promoting motifs beyond a few conventional motifs, or about how proteins work together in condensates to achieve their biological function. We propose a research program that systematically elucidates novel motifs and biophysical principles in protein- only condensates. This will be accomplished by leveraging innovative approaches such as CRISPR imaging, optogenetics manipulation, and custom-written analysis and quantification algorithms to pursue two interrelated research themes. The first theme is identification of alternative mechanisms that enable protein condensate formation by: discovering novel condensate-promoting motifs by interrogating fusion proteins known or suspected to form condensates; determining the essentiality and modularity of such motifs; and mapping motif- function relationships in condensate-mediated processes. The second theme is to uncover physical principles that regulate composition and dynamics in multi-protein condensates. Although the molecular details may differ, the sequence space and principles demonstrated here are broadly applicable to a diverse range of proteins and cellular processes. The long-term objective of our research program is to build an expanded biophysical foundation to understand biomolecular condensate assembly and functions across cellular homeostasis and pathology. Such knowledge brings new opportunities to modulate cellular processes through independent physical approaches instead of traditional ways of interfering with biochemical reactions, and provide a biophysical framework to prevent, diagnose, and treat condensate-driven diseases.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY (See instructions): The emergence of single-cell RNA-sequencing (scRNA-seq) techniques has motivated many computational methods to study gene regulation and cell differentiation at the single-cell level. However, to improve the translational value of scRNA-seq, new methods are required to comparatively study the molecular differences between normal and pathological cells/tissues, and between control and case/treatment groups. The vast majority of existing network and clustering models have focused on scRNA-seq data generated under one experimental condition. Moreover, most existing scRNA-seq network models are correlative in nature and do not infer causality. What remains lacking are rigorous statistical methods for inference of differential causal gene regulation and cell composition in response to experimental interventions. There is, therefore, a critical need to develop novel methodologies for identifying the effects of experimental interventions on causal gene regulatory relationships and cell differentiation by jointly modeling scRNA-seq data across experimental groups. Without such tools, mechanistically understanding gene regulatory activities and cell differentiation will likely remain difficult. Our overall objective is to design and validate Bayesian network and clustering models for identifying differential causal gene regulatory networks and cell composition for scRNA-seq data generated under different experimental conditions. To achieve the overall objective, three specific aims will be pursued: Aim 1: Develop a differential zero-inflated negative binomial Bayesian network model to construct differential cell-type-specific causal gene regulatory networks under two experimental conditions. Aim 2: Develop a trajectory-dependent directed cyclic graph model to construct cell-specific causal networks with feedback loops and monitor its structural changes with cell development. Aim 3: Develop a scalable Bayesian semiparametric differential clustering method to discover differential cell composition and cell-type-specific marker genes that are shared and/or unique to each experimental group.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY This R01 application is responsive to the NIH initiative PAR-19-253 “Focused Technology Research and Development”. Assays for measuring receptor-ligand affinity are valuable in many areas of biomedical research. The “gold standard” surface plasmon resonance assay is limited to recombinant soluble receptors fixed on solid surfaces. The emerging adhesion frequency assay (AFA) techniques can measure the receptor-ligand affinity on their native cellular membranes. However, existing AFA methods can neither resolve the non-uniform distribution of receptors on single cells nor measure the rolling cell adhesion under shear forces. In addition, currentAFAapproaches are generally bulky and low throughput, which require tedious operation. Recently, we have invented a light-driven microrobot (LDM) platform as a non-invasive, programmable, and multimodal cell-manipulation technology. Based on this versatile LDM platform, we propose to develop a paradigm- shift four-dimensional (4D) AFA (i.e., integrated 3D translational AFA and 3D rotational AFA) to overcome these key obstacles in the existing assays. In this R01 project, we will develop and validate our 4D AFA with the following features: (1) measuring receptors on their native cell membrane environments, (2) resolving the non-uniformly distributed receptors on single cells, (3) enabling both translational and rotational AFAs on an integrated platform, (4) investigating cell adhesion under both shear force and tensile force, and (5) allowing on-chip multiplexed cell adhesion measurements. With such features, the proposed 4D AFA has the potential to exceed current lab standards, address unmet needs in the field, and enable high-throughput full profiling of receptor-ligand interactions at sub-cellular resolution. We will validate and improve the 4D AFA performance using well-studied receptor-ligand pairs with variable affinities. We will further package and apply the validated assay to investigate the binding of SARS-CoV-2 virus to angiotensin-converting enzyme 2 receptor and to screen T cells for immunotherapy for cytomegalovirus infection. In this regard, we aim to demonstrate the far-reaching potential of 4D AFA to enable improved research in areas ranging from clinical immunotherapy to fundamental biology.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT A major risk factor for developing alcohol use disorder (AUD) is a reduced level of response to alcohol. In persons with normal alcohol metabolism, differences in level of response to an acute intoxicating dose of alcohol result mainly from differences in acute tolerance to alcohol. Tolerance has a major influence on alcohol consumption; tolerance to alcohol’s rewarding effects encourages more drinking to achieve a desired effect, whereas tolerance to alcohol’s aversive properties reduces a disincentive to drink. This proposal is based on recent findings that inhibitors of phosphodiesterase 4 (PDE4) reduce alcohol drinking in rodents. In our work with the PDE4 inhibitor apremilast, we were struck by the relationship between its ability to reduce both alcohol tolerance and ethanol consumption. Inhibitors of PDE4 reduce metabolism of cAMP which leads to activation of protein kinase A (PKA). Alcohol has prominent effects on GABAA receptors, and it has been known for many years that PKA alters the function of GABAA receptors through phosphorylation of β1 and β3 receptor subunits. Our preliminary experiments led us to our overall hypothesis that PDE4 inhibition reduces alcohol tolerance and alcohol consumption by increasing GABAA receptor function in the brain through PKA-mediated phosphorylation of GABAA receptor β3 subunits, with PKA-mediated phosphorylation of β1 subunits contributing a minor opposite effect. Studies are planned to test this hypothesis by determining whether apremilast increases PKA-mediated phosphorylation of b3 and β1 subunits in hippocampal slices from wild type mice. Studies will examine the role of PKA phosphorylation on alcohol-related behaviors using b3- S408A/S409A and b1-S409A knock-in mice, which lack the PKA phosphorylation sites of interest. We will examine the role of specific brain PDE4 isozymes in these alcohol-related behaviors using PDE4 isozyme- selective inhibitors and knockout mice. We will also determine whether b3 and β1 subunits associate with specific PDE4 isoforms by isolating GABAA receptor complexes using Blue Native PAGE and analyzing them with mass spectrometry (LS-MS/MS). Finally, we will test the hypothesis that a b1-selective, positive allosteric modulator (PAM) alone or in combination with apremilast will reduce alcohol tolerance and alcohol consumption. These experiments will characterize a new series of 10 novel compounds targeting b1-containing GABAA receptors using receptors expressed in Xenopus oocytes, to identify a b1-selective PAM with properties suitable for testing in mice.

NIH Research Projects · FY 2025 · 2022-09

Up to 60% of breast cancer survivors report chronic pain that interferes with daily activities and is frequently associated with negative health outcomes such as fatigue, sleep disturbances, and decreased quality of life. Especially, those with depressive symptoms are more likely to have inadequate pain management due to their depression, and pain could further deteriorate depression. Furthermore, the recent opioid crisis has exacerbated their fear of addiction and reluctance to seek help for pain management. However, survivorship programs hardly address these needs of survivors who have transitioned to communities while considering their unique needs. A technology-based approach using computers and mobile devices promises to meet the needs with high flexibility, accessibility, and anonymity. Based on Preliminary Studies (PSs), the research team developed and pilot-tested an evidence-based Web App-based information and coaching/support program for cancer pain management (CAPA) using multiple unique features. However, CAPA rarely considered depressive symptoms accompanying pain in its design or components, and PSs indicated the necessity of further individualization of the intervention components of CAPA to address the unique needs of these survivors. The purpose of the proposed 2-phase study is to further develop CAPA with additional components for breast cancer survivors with depressive symptoms and the individual optimization functionality (CAI) and to test the efficacy of CAI in improving cancer pain experience. As the initial group to approach, Asian American breast cancer survivors with depressive symptoms (ABD) were selected as the target population, given their relatively higher rates of inadequate pain management and the associated lower quality of life compared to other groups. The specific aims are to: a) develop and evaluate CAI through an expert review and a usability test (R61 phase); b) determine whether the intervention group (that uses CAI and usual care) will show significantly greater improvements than the active control group (that uses CAPA and usual care) in primary outcomes (cancer pain management and cancer pain experience including depressive symptoms) from baseline to post 1-month and post 3-months; c) identify theory-based variables (attitudes, self-efficacy, perceived barriers, and social influences) that mediate the intervention effects of CAI on the primary outcomes; and d) determine whether the effects of CAI on the primary outcomes are moderated by selected background, disease, genetic, and situational factors. This study is guided by the Bandura’s Theory and the stress and coping framework by Lazarus and Folkman. The R61 phase includes: (a) the intervention development process, (b) a usability test among 15 ABD, 15 family members, and 15 community gatekeepers; and (c) an expert review among 10 experts. The R33 phase adopts a randomized repeated measures control group design among 300 ABD. Long-term goals are to scale the program across various settings and among the general U.S. population while advancing innovations and frameworks for highly customized technology-based interventions designed for cancer survivors.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Acute trauma, defined by the DSM-5 criterion A for the diagnosis of post-traumatic stress disorder (PTSD) as exposure to actual or threatened death, serious injury or sexual violence, is associated with an increase in the development of both Alcohol Use Disorder (AUD) and PTSD. It is well established that patients with PTSD have a markedly increased risk for AUD. Furthermore, understanding risk for AUD during development, particularly in adolescents is critical, as early onset drinking is one of the highest risk factors for lifetime alcohol addiction. This current proposal seeks to further explore the relationship of AUD and PTSD in adolescents exposed to acute trauma by focusing on biological predictors of AUD and/or PTSD development. In brief, 500 adolescents will be studied in the immediate aftermath of trauma in emergency departments, hospital clinics or psychiatric settings in Austin, Texas (Dell Children’s Medical Center) and Galveston, Texas (University of Texas Medical Branch (UTMB) affiliated hospitals). Using emerging statistical techniques and machine learning-based analytics, we will identify predictive: 1. Genomic and epigenomic, 2. Inflammatory, and 3. Psychophysiological biomarkers of risk for AUD and PTSD. Although several risk factors have been identified in adults for the development of these two disorders, relatively little data is available in adolescents. Family and twin studies have provided estimates of genetic risk for AUD and PTSD of 50% and 30-40%, respectively. We will utilize the latest AUD and PTSD polygenic risk factor scores for these disorders, together with epigenomic analysis. Previous work has implicated alterations in inflammatory response in both AUD and PTSD and we will assess this in the immediate aftermath of the trauma. Finally, measures of autonomic nervous system (including skin conductance response [SCR], heart rate and heart rate variability [HRV] via electrocardiography [ECG]) and central nervous system (acoustic startle response assessed via electromyography [EMG]) reactivity will be assessed immediately after post-trauma medical clearance. Using state of the art statistical modeling, we will identify biological predictors of AUD and PTSD and their interrelationship. These data will be used to develop novel tools to predict which adolescents exposed to trauma will likely develop AUD and/or PTSD, allowing for early intervention at this critical time in development.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT DNAzymes represent one of the most recent classes of diverse, catalytically active biomolecules. However, despite their discovery >25 years ago and exceptional potential for broad analytical and therapeutic applications, our understanding of metallo-DNAzymes in terms of binding selectivity, structure, and catalytic mechanism still lags far behind that of metalloproteins. Although DNAzymes have already been developed as highly selective metal ion sensors, the lack of fundamental knowledge regarding metallo-DNAzyme function has precluded the application of rational design to enhance metal binding affinity and specificity. The goal of this project is to obtain unparalleled insight into the structure-function relationships of metal-binding DNAzymes specific for redox-active metal ions (RAMIs) with high physiological relevance (e.g., Fe2+, Fe2+, Cu+, and Cu+2), and in turn provide a foundation for the future rational design of DNAzymes. To achieve this goal, an array of biochemical and advanced biophysical characterization techniques will be employed and cross-correlated to determine the locations of metal binding, coordination environments, binding affinities and specificities, and reaction mechanisms for a series of Fe2+, Fe3+, Cu+, and Cu+2-specific DNAzymes. Metal-bound DNAzyme resting states will be generated using a series of “non-cleavable” substrates, which will prove fundamental in determining metal binding affinities, specificities, and key spectroscopic signatures using UV-Vis/nIR, EPR, and 57Fe Mössbauer spectroscopies. By additionally applying XAS, the rudimentary coordination environment and electronic structure will be determined. Single point mutations will be screened across suspected metal-binding regions of oligonucleotide sequences, and the corresponding cleavage efficiency and characteristic spectroscopic signatures will be tracked to narrow the assignment of metal-binding site. Further advanced characterization using vibrational and pulse EPR spectroscopies will be used together with selectively isotope-labeled residues to provide a precise assignment of metal binding location and coordination environment. All of this information will be matched by computational modeling of first coordination binding models using a DFT and ab initio approaches. Lastly, a high-risk/high-reward foray will be made to grow diffraction-quality crystals for holistic structural characterization. Beyond the resting state, the mechanism of DNA/RNA cleavage by these DNAzymes will be analyzed by a combined analysis of cleaved fragment ends and careful kinetic characterization. Where necessary, rapid quench flow and rapid freeze quench methods will be employed to trap and assess potential reaction intermediates. Achieving the above goals will greatly deepen our understanding of the structure and function of metal- binding sites in DNAzymes, shifting the paradigm of metalloprotein characterization methodology to include metallo-DNAzymes. These insights are crucial for rational design and computational modeling to be used effectively in producing the next generation of metal ion-sensing DNAzymes.

NIH Research Projects · FY 2025 · 2022-09

Summary/Abstract: Our long-goal is to develop an unprecedented semi-autonomous surgeon-in-the-loop surgical robotic system and complementary computer-assisted algorithms to enable an intuitive in situ robotic bioprinting of human tissues and organs. More specifically, using this extrusion-based bioprinting system, a surgeon can (i) first utilize a high-resolution three-dimensional (3D) point cloud camera to plan an arbitrary spatial printing geometry on the target anatomical surface, (ii) co-operate with a robotic system to manipulate a custom- designed bioprinting instrument to precisely follow the planned printing geometry, and (iii) perform an intuitive and precise deposition of engineered bioinks to make tissue constructs on the target anatomical surface, while (iv) directly control and monitor the printing process to ensure the safety and success of the procedure. The focus of this proposal is simultaneous functional and cosmetic restoration of large volumetric muscle loss (VML) injuries by utilizing a novel engineered bioink- developed by our collaborators at the Terasaki Institute of Biomedical Innovation, a complementary robotic bioprinting system, and intuitive computer- assisted algorithms. Severe musculoskeletal injuries can lead to VML, where extensive musculoskeletal damage and tissue loss result in permanent loss of function. In small-scale injuries or strains, muscle is capable of endogenous regeneration and complete functional restoration. However, this ability is abated in VML, where the native biophysical and biochemical signaling cues are no longer present to facilitate tissue regeneration. Current state- of-the-art in vitro tissue engineering VML treatment procedures suffer from various issues including (i) prolonged culturing period in bioreactors demanding functionality enhancement prior to implantation in the body; (ii) adhesion failure of in vitro 3D printed hydrogel scaffolds to the remnant muscle, whether injected, sutured, or placed into the wound; and (iii) inability to be printed precisely in irregular curved 3D surfaces of large VML injuries. It is our central hypothesis that the proposed semi-autonomous robotic bioprinting system can collectively address the mentioned limitations of the current state-of-the-art solutions by (i) reducing complexity, surgical time, and complications associated with current VML treatments, (ii) immediately delivering and in situ printing of appropriate bioinks to the target anatomy and utilizing the human body as a natural bioreactor to induce tissue maturation and function, and (iii) providing real-time feedback on the 3D bioprinted constructs as well as the surgeon’s and patient’s motions to ensure precision of the bioprinting procedure for simultaneous functional and cosmetic restoration of the injured muscle. The proposed project is multidisciplinary and bridges the current gap between the robotic surgery, tissue engineering, and bioprinting fields. The contribution is significant, high impact, and innovative and can revolutionize the current clinical paradigm.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Elevated risk taking contributes not only to the development of substance use disorder (SUD) but also the likelihood of relapse. There has been significant progress in delineating the neural substrates underlying this causal relationship. Nonetheless, we are still faced with a significant barrier in translating these findings to the clinical setting because the majority of the work on this topic has used male subjects. This is despite the well- established sex differences in risk taking and aspects of SUD. This significant limitation in scientific advancement can be remediated by examining neurobiological mechanisms underlying decision making in females. The long-term goal of our lab is to identify the neural mechanisms mediating risk taking in females and how hormones contribute to these processes. To meet this goal, I will use a rodent model of risk taking in which females are more risk averse and exhibit greater sensitivity to risk of punishment than males. In this model, female risk aversion is largely mediated by estradiol (E2) and such E2-dependent risk aversion requires estrogen receptor (ER) β. We have also established a role for the basolateral amygdala (BLA) and its projections to the nucleus accumbens (NAc) shell in promoting risk averse behavior. Preliminary data reveal that activation of D2 dopamine receptors (D2Rs) in the BLA increases risk aversion in females, but not males. These findings suggest that differences in BLA function may underlie sex differences in risk taking, and specifically, promote risk aversion in females. Prior work shows sex differences in BLA-dependent behavior are due to the ability of E2 to modulate BLA activity and function. Given the role of the BLA in risk taking and the fact it is potently modulated by E2, it is therefore conceivable that risk aversion in females may be due to E2 regulation of BLA activity necessary for risk-based decision making. Consequently, the overall objective of this proposal is to dissect the neural mechanisms by which E2 promotes risk aversion in females. I hypothesize that E2 mediates female risk aversion through its modulation of ERβ and D2R function in the BLA and its projections to the NAc shell. I will test my hypothesis by carrying out three experimental aims. In Aim 1, I will identify the contributions of ERs in the BLA to E2-dependent risk aversion in females using RNA interference-mediated ER gene reduction. In Aim 2, I will identify the necessity of E2 modulation of D2R function in the BLA for risk aversion in females using optogenetic manipulation of BLA neurons that selectively express D2Rs. In Aim 3, I will identify the contribution of E2 modulation of BLA projections to the NAc shell to risk aversion in females using optogenetic manipulation of this circuit. This project is significant because the knowledge gained will advance our understanding of the neural mechanisms by which E2 mediates female risk aversion and provide a foundation from which we can determine whether a disruption in these processes contributes to elevated risk taking associated with SUDs.

NIH Research Projects · FY 2025 · 2022-09

Project Summary 6.2 million Americans are living with Alzheimer’s disease (AD), experiencing reduced quality of life and irreversible deterioration of cognitive function. AD development is likely driven by inflammation in the central nervous system (CNS), yet there remain no broadly effective therapies. We propose that AD pathology may be, in part, caused by circadian dysregulation of peripheral immune cell migration through the CNS. The circadian clock is a critical regulator of biological processes, generating ~24 h rhythms in gene expression, hormone release, and behavior, but its efficacy in various cells and tissues deteriorates with age. Up to 70% of individuals with AD experience circadian disruption and sleep-wake disturbances, which often present years before clinical diagnosis. Circadian dysregulation is therefore a potential biomarker and signal for early intervention in age- and AD-related pathology. The immune system is tightly regulated by the circadian clock, generating daily cycles of immune cell migration throughout the body which leads to temporal windows of high and low immune reactivity. The immune system also critically regulates CNS function; peripheral inflammation disrupts behavior and impairs cognition. During aging, the CNS immune system gradually shifts from a balance between pro- and anti- inflammatory function towards a more reactive inflammatory state. Importantly, trafficking of adaptive immune cells to immune-brain interfaces regulates neuroinflammation and cognition. Thus, we hypothesize that circadian rhythms in immune cell trafficking to the CNS are disrupted with age and AD-like pathology, leading to cognitive and behavioral changes. This proposal addresses the following specific aims: First, establish the role of circadian dysregulation of immune cell trafficking in CNS inflammation and cognitive decline during aging and AD-like pathology; second, determine if disruption of molecular clocks in key brain-immune interface cells expedites cognitive decline during aging and AD-like pathology; and third, reveal whether reinstating daily trafficking rhythms via time-restricted feeding ameliorates age- and AD-induced pathology. This proposal is innovative in combining expertise in circadian biology, immunology, and behavioral neuroscience to understand how circadian regulation of brain barriers and immune cell trafficking through the CNS regulates aging and AD-like pathology. This contribution will be significant because our results will identify how circadian regulation of adaptive immune cell migration through the CNS affects local neuroimmune function. We expect that our results will identify a novel role for biological rhythms in regulating neuroinflammatory pathology and cognition via immune cell migration, highlighting novel therapeutic avenues to target cognitive decline and behavioral changes in aging, AD, and AD-related dementia.

NIH Research Projects · FY 2025 · 2022-09

7. PROJECT SUMMARY The 60 million rural dwellers across the United States are older, more likely to live in poverty, and more likely to be either underinsured or uninsured compared to their urban counterparts. Moreover, the 240,000 rural patients with end stage kidney disease (ESKD) have less access to nephrology care, are less likely to finish pre-kidney transplant evaluation, and are less likely to undergo kidney transplantation. Telehealth is uniquely positioned to overcome geographic barriers of rural America by capitalizing on electronic information and telecommunication technologies. Yet telehealth is underutilized among rural patients in general, and little is known about how geography, resources, and distance to healthcare facilities contribute to access to care, outcomes, and quality of life for rural patients with ESKD. A comprehensive study of utilization, cost-effectiveness, and patient and provider preferences would be an important step in expanding telehealth policies specifically aimed to care for the rural ESKD population. We propose the following specific aims: (1) to assess the costs, utilization, and outcomes associated with telehealth for rural patients with ESKD; (2) to compare telehealth provision of ESKD care to standard in-office care in the rural ESKD population using model-based cost-effectiveness analysis; and (3) to understand facilitators and barriers of using telehealth from the perspective of patients and providers. A detailed training plan for Joel T. Adler, MD, MPH, is outlined in this proposal. In brief, it includes in-depth coursework to extend and expand Dr. Adler’s research skills, a practicum experience with the RURAL (Risk Underlying Rural Areas Longitudinal) Cohort study in the southern rural United States, and a mentorship plan jointly prepared by the principal investigator and a team with expertise in kidney disease, health services research, qualitative research, cost-effectiveness analysis, and the mentorship of junior clinician-scientists. This will help the candidate meet the following career goals: (1) gain state of the art expertise in large claims database, geographic information systems, cost-effectiveness, and qualitative research, (2) apply for and obtain R01 grant funding, and (3) transition to academic independence. When completed, Dr. Adler will have learned the perspectives of rural patients with EKSD and providers on telehealth that will be methodically crucial in designing pilot studies that inform R01 proposals to increase accessibility and utilization of telehealth on a population-health level for rural patents with ESKD. These efforts will culminate in a comprehensive understanding of the role of telehealth in caring for rural patients with ESKD by economic assessment of its utilization, a cost-effectiveness analysis for implementation on a wider scale, and learning how these align with patient and provider preferences to inform future policy regarding telehealth utilization and reimbursement.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Spatial and temporal heterogeneity in the cellular environment has profound implications in biological processes related to human health/disease. Many single-cell analytical tools have been developed over the years to reveal the heterogeneity among cells, e.g., the spatial distribution of chemicals and ions. However, one missing piece in the single-cell analysis is the ability to reveal quantitatively the spatial and temporal heterogeneity cellular response to chemical stimulus. This is challenging because controlling the exact concentration of chemicals at a specific location depends on the interplay between dynamics of mass transport in the complex cellular environment and the reactivity of the molecules. Indeed, some physiologically important molecules, including reactive oxygen species (ROS), reactive nitrogen species (RNS), are highly reactive and have short lifetimes. A tool for precision delivery of molecules, including these reactive ones, are necessary to quantitatively study their effect at the single-cell level. Our research lab will focus on developing nanoscale precision delivery tools to quantitatively control the delivery of molecules of biological interest, including those highly reactive ones. The strategy is based on a functionalized nanopipette electrode that is capable of in situ generation of the molecule of interest electrochemically with spatial and temporal control. This will be demonstrated by the delivery of nitric oxide (NO), a reactive molecule whose transient concentration is important in neuron transmission, immune response, and blood coagulation. Spatial and temporally resolved delivery is achieved by combining the electrochemical chemical delivery system with nanoscale electrochemical imaging techniques. This delivery modality can be extended to other reactive molecules, including H2S, CO, and ROS. In addition, we will develop a precision delivery tool called digital delivery, where we will precisely control the number of biomolecules or other non- biological entities being delivered, including proteins and nanoparticles, by counting their number during the delivery in a resistive pulse fashion. Lastly, we will quantitatively map the spatially resolved rate of uptake of the molecules being delivered. Ultimately, the precision delivery methods developed in our proposed research will enable quantitative investigation of many fundamental biological and physiological questions related to the reactive molecules at the single-cell level. For example, the tools can be used to reveal the spatial and temporal heterogeneity in the neuron response by precision delivery of neuron transmitters or their vesicles. The modality can also be applied to quantitatively modulate or stimulate the inflammatory response at the single-cell level.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT Excessive alcohol use is driven by various factors that are biological, psychological, and social in nature. The degree of how these factors contribute to problematic drinking and then relapse after recovery vary widely at the individual level. One common factor presented to all the users of alcohol is the set of cues closely linked to alcohol consumption. Environmental cues that reliably precede alcohol availability and subsequent consumption can gain the ability to elicit conditioned responses such as alcohol-seeking behavior and promote problematic drinking. Current treatment for alcohol use disorder includes cue-exposure therapy, a behavioral procedure in which alcohol cues are systematically presented in the absence of alcohol to promote a reduction or “extinction” of cue-conditioned responses. Unfortunately, extinction is rarely permanent: extinguished cue-conditioned alcohol-seeking responses are highly susceptible to relapse. We recently showed that a modification to standard extinction procedure (i.e., cue-induced memory retrieval session prior to extinction) blocked the return of alcohol- seeking behavior of rats with moderate level of alcohol consumption (Cofresi et al., 2017). This “retrieval+extinction” approach has enormous potential in that a simple modification to cue-exposure therapy protocols used in alcohol rehabilitation could significantly improve the odds of preventing relapse following treatment. It is thus crucial to extend our initial finding to a rat model of alcohol dependence that better mimics the human conditions of alcohol abuse. In fact, no study has systematically examined the nature and mechanism of extinction, let alone retrieval+extinction, in a rat model of alcohol dependence. Therefore, we will determine parameters that influence extinction and relapse of alcohol-seeking behavior in dependent rats, and further examine whether retrieval+extinction is equally effective at preventing relapse in dependent rats as it was in non- dependent rats. Then, we will probe the neural substrates underlying extinction of alcohol-seeking behavior in dependent rats and test the hypothesis that retrieval+extinction engages neural mechanisms that are distinct from standard extinction.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY. Recent epidemiological reports indicate that cocaine use increased by 45% from 2013 to 2018. Cocaine-induced increase in striatal dopamine levels is linked to its rewarding effects. However, the striatal neural circuits driving cocaine abuse are not clearly defined. Thus, there is a critical need to delin- eate how the striatal circuits downstream of cocaine-induced dopamine release control cocaine seeking and taking. The overall objective of this proposal is to determine the mechanisms by which cocaine affects GABA transmission between the two principal neurons of the striatum, the D1-MSNs and D2-MSNs, to ultimately regulate drug seeking and taking. Our central hypothesis is that cocaine-enhances levels of the opioid peptide enkephalin, which acts via mu-opioid receptors (MOR) expressed in axon collaterals of D1- and D2-MSNs to facilitate cocaine seeking and taking. We will test this in two specific aims: Aim 1: Determine the mechanisms for how cocaine affects plasticity of intra-striatal GABA trans- mission. Based on our preliminary data, we hypothesize that a history of cocaine increases enkephalin re- lease from D2-MSNs, which induces MOR-dependent long-term depression of intra-striatal GABA transmis- sion onto D1-MSNs. We will test this by performing whole cell electrophysiology in MSN-selective MOR and enkephalin knockouts with a history of cocaine or saline exposure, and record GABA transmission between MSNs and MSN excitability. Pharmacology will be used to infer heightened enkephalin tone in electrophysi- ology recordings, and this will be confirmed by RNAscope, immunohistochemistry, Western blot and our MSN- selective enkephalin knockout. Aim 2: Determine how opioid regulation of intra-striatal circuits drive cocaine seeking and taking. Based on our preliminary data we hypothesize that enkephalin released from D2-MSNs acts on MORs in axon collaterals of D1- and D2-MSNs to suppress intra-striatal GABA and facilitate cocaine reward. We will test this using operant cocaine self-administration procedures in MSN-selective MOR and enkephalin knock- out mice. We will determine how intra-striatal GABA transmission from MSNs contributes to cocaine seeking by selectively inhibiting D1-MSN or D2-MSN striatal axon collaterals using a novel Gi-coupled opsin during a cued cocaine seeking task. Successful completion of the proposed research will elucidate how endogenous opioids regulate intra- striatal GABA transmission, and how cocaine impinges upon this mechanism to affect circuit activity. Moreo- ver, this research will provide novel insights on the mechanisms of cocaine abuse by establishing a link be- tween opioid-mediated regulation of intra-striatal GABA transmission and cocaine seeking and taking. This research will also lay the groundwork for our long-term goal of determining the circuit mechanisms driving synergistic reward when opiates and cocaine are co-abused.

NIH Research Projects · FY 2025 · 2022-08

Project Abstract Over the past decade, optogenetics has increasingly become an important technology for spatiotemporal control of neural activity, cardio functions, muscle cell activity, protein-protein interaction, and disease applications, through the genetically encoded light-activated proteins. However, there are still two major challenges of this technology: 1.the delivery of light to into deep body areas such as brain or heart generally requires the optical fiber implantation which could result in damage of cells and tissue. 2. Gene expression requires viral transduction, which suffer from a number of limitations such as the host immune response, the stability expressed proteins over time, the limitations on maximum gene size and the lack of economic scalability for manufacture. To address the first challenge, we recently developed a technology named ‘sono-optogenetics’ to convert focused ultrasound (FUS) to light for non-invasive optogenetics. The nanoparticles are injected into the circulating blood so that neither craniotomy nor intracranial implantation is required for achieving optogenetics. However, these inorganic nanoparticles are generally difficult to be modified to emit different colors of light for multiplex optogenetic control and are not biodegradable after accumulating in the animal livers after use, causing long-term safety concerns. Therefore, the goal of this proposal and the focus of my research lab, is to tackle the remaining challenges for optogenetics through designing organic nanomaterials, including hydrogen-bonded organic frameworks nanoparticles, chemical assembly of DNA plasmids and cationic polymer delivery agents. Specifically, we are planning to 1) design biodegradable nanoparticles to convert ultrasound to light for multi-colored sono- optogenetics. 2) improve the delivery of plasmid DNAs through nucleopore through covalent chemical assembly strategies and 3) design advanced cationic polymers for improving endosome escape, cellular uptake and diffusivity through extracellular space in non-viral gene delivery. The work will also enhance our understanding the transport and interaction of organic nanoparticles in cells. As a result, I believe that the proposed works is well suited for the NIH R35 Maximizing Investigators’ Research Award.

NIH Research Projects · FY 2025 · 2022-08

Abstract Mohs micrographic surgery (Mohs) is the most effective method to treat nonmelanoma skin cancer. Mohs achieves high success (98% cure rates) by assessing surgical margins intraoperatively with frozen section histopathology. Unfortunately, the sophisticated infrastructure and laborious process needed to perform frozen section histopathology leads to lengthy, expensive surgeries that limit access and result in disparities of care. We propose to develop "optical Mohs" as a rapid, low-infrastructure alternative for Mohs-indicated patients in rural and other underserved populations who do not currently undergo Mohs surgery. Our optical Mohs approach will be based on multimodal confocal microscopy (MCM) combined with machine learning to provide a low infrastructure, automated diagnostic tool requiring minimal tissue processing. MCM combines reflectance, fluorescence, and Raman confocal microscopy into a single benchtop platform. MCM (using reflectance and fluorescence) has recently demonstrated success in producing H&E images of unprocessed, freshly excised skin that pathologist can read with accuracy comparable to frozen section histopathology. However, this approach alone still requires a pathologist to read the image. Machine learning is being explored to automate the diagnosis of these images, but has not yet yielded sufficient accuracy. We hypothesize that the addition of Raman spectroscopy will significantly increase the diagnostic accuracy of an automated approach. Raman is a complementary approach that is sensitive to the skin’s molecular composition and has been proven in clinical margin detection studies within the skin with sensitivities of 92-100% and specificities of 84-93%; however, a critical barrier to its adoption has been its slow acquisition speed. We introduce two innovations in Raman acquisition (superpixel and line scanning) that enable acquisition of Raman at speeds compatible with surgical guidance (speeds of 1cm2/min.). Our preliminary model in thirty patients demonstrates that a predictive model trained on both structural reflectance confocal images and biochemical information extracted from Raman images discriminates basal cell carcinoma from normal structures with very high accuracy, suggesting that optical Mohs could help dermatologists "keep cutting" as needed to remove the entire tumor (100% sensitivity) while not removing an excessive amount of healthy tissue (92% specificity). We will design, fabricate and bench-test an MCM instrument (Aim 1). We will design a decision-support system for tumor margin assessment based on MCM images using a post-surgery data set in 108 patients (Aim 2). We will determine the accuracy of the decision support system for tumor margin assessment based on MCM imaging in an intraoperative setting in 72 patients (Aim 3). The potential clinical outcome would demonstrate that an optical Mohs guided surgery could be used where conventional Mohs is indicated but not currently used, expanding access of Mohs’ accuracy to populations currently not receiving his level of care.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract. Fragile X syndrome (FX) is a widespread type of inherited intellectual disability. Effective treatments that target mechanisms underlying FX are currently lacking. FX is the foremost monogenic cause of autism spectrum disorders, and thus many individuals with FX exhibit abnormal social behaviors. Individuals with FX also often engage in aberrant spatial behaviors such as “elopement”, wandering off and getting lost. The hippocampus is a brain structure that is particularly vulnerable to FX. Much evidence suggests that hippocampal areas CA2 and CA1 are important for social behaviors and spatial memory, respectively. Yet, few studies have investigated whether disturbances in neurophysiological mechanisms in CA2 and CA1 could underlie impaired social and spatial cognitive functioning in FX. This project’s goal is to address this gap in knowledge by investigating the extent to which subcellular, cellular, circuit, and neuronal population mechanisms of social and spatial memory operations in the hippocampus are impaired in rodent models of FX. The studies will employ state-of-the-art in vivo and in vitro electrophysiological techniques. In vivo approaches will be used to assess whether impairments in cellular responses in CA2 and coordinated neuronal population activity in CA1 could explain deficits in social and spatial cognition in FX. In vitro experiments will be conducted to uncover cellular mechanisms underlying altered intrinsic properties of and plasticity in CA2 neurons and aberrant inhibitory circuits in CA1. Models of FX in two species, specifically Fmr1 knockout (KO) rats and mice, will be used, allowing comparison of FX pathophysiology across species. Specific Aim 1 will assess whether correlated neuronal spiking activity between CA2 and one of its major inputs, CA3, is weaker in Fmr1 KO rats than wildtype rats during exploration of social stimuli. Specific Aim 2 will employ whole cell and patch clamp recordings, including recordings directly from dendrites, in hippocampal slices to test whether CA2 neurons in Fmr1 KO rats and mice show impaired synaptic plasticity and deficient responses to the social neuropeptide, oxytocin. Specific Aim 3 will test whether reactivation, or “replay”, of spike sequences from populations of CA1 neurons that code for previously learned spatial trajectories is disrupted in Fmr1 KO rats. Replay is critical for spatial memory operations, and thus disrupted replay could contribute to impaired spatial cognition and behavior in FX. Replay of CA1 neuronal spike sequences is temporally coordinated by properly timed activation of specific CA1 inhibitory interneurons. Thus, disrupted replay of CA1 spike sequences in FX may reflect disturbances in CA1 inhibitory circuits. Specific Aim 4 will employ whole cell recordings from CA1 pyramidal neurons, specific classes of CA1 interneurons, and connected CA1 interneuron-pyramidal cell pairs to test the hypothesis that inhibitory circuits are disrupted in FX. Successful completion of these Aims will provide novel insights about specific mechanisms underlying aberrant social and spatial cognition and behaviors in FX. Gaining a deeper understanding of FX mechanisms is expected to suggest novel targets for intervention in FX.