Trustees Of Indiana University

NIH Research Projects · FY 2026 · 2026-06

Despite the proven efficacy of non-invasive behavioral and pharmacologic guideline-directed medical treatments (GDMTs) for peripheral artery disease-related claudication, their clinical implementation remains suboptimal. Within a year of diagnosis, 34% of patients have one or more major adverse vascular event (e.g., stroke, heart attack, amputation) which are largely preventable with first-line GDMT prescription. However, data indicate GDMT is underprescribed in real-world clinical practice and there is patient-level variation in prescriptions and outcomes. While these data imply that patient-level factors may influence GDMT prescription and outcomes, previous research has relied on structured data sources, limiting our ability to quantitatively characterize the full spectrum of these factors. An additional data-related challenge has been our ability to identify behavioral GDMTs, which are often recorded in the unstructured clinical notes. Recent advancements in digital health and generative artificial intelligence-based large language models (LLMs) have made it possible to extract and convert these unstructured qualitative EHR data into quantified structured data for analysis. Leveraging these advancements has the potential to improve the use of GDMT for peripheral artery disease-related claudication and to improve outcomes in the nearly 10 million people with claudication. Our long-term goal is to achieve optimal treatment outcomes in the largest number of individuals with peripheral artery disease-related claudication. Our main objective is to produce a validated BehavioralLLM that will identify the prescription of behavioral GDMTs. Our secondary objective is to characterize the influence of patient-level factors on GDMT prescription and the mediating influence of GDMT on poor treatment outcomes experienced by people with claudication. We will achieve our objectives via the following aims: 1) Optimize and validate a BehavioralLLM to extract and identify behavioral GDMT prescription information; 2) Establish the influence of patient-level factors on all GDMT prescription for claudication; 3) Evaluate the mediating influence of GDMT prescription concordance on the relationship between patient factors and treatment outcomes. In aim 1, we will use generative AI to identify prescription of behavioral GDMTs from unstructured EHR data. In aim 2, we will leverage robust data on patientlevel factors and prescription of GDMTs using structured and unstructured EHR data. In aim 3, we will determine outcome differences that can be reduced under counterfactual scenarios where GDMT prescription concordance is maximized. The expected outcomes include a validated ‘BehavioralLLM’ used to identify the behavioral GDMT prescriptions and characterization of the influence of patient-level factors on GDMT prescription and the mediating influence of GDMT on poor treatment outcomes experienced by people with claudication.

NIH Research Projects · FY 2026 · 2026-05

Project Abstract This proposal aims to develop statistical models that associate brain connectivity with human health outcomes. It uses a mathematical framework that quantifies not only pairwise co-activation of brain regions (nodes), but also encodes three-way and higher-order interactions, and their densities, using the mathematical framework of simplicial complexes (SCx). The methods developed here will enable the statistical analysis of cognitive function in large neuroimaging studies by modeling connectivity patterns in ways that are more extensive than those currently used. These methods will provide new insights into the complexities of brain-related health conditions because they quantify neuro-activation patterns in new and interpretable ways. Aim 1, extends the investigators’ previous scalar-on-matrix regression to include generalized linear and mixed models, then moves beyond adjacency matrix predictors to upper-adjacency edge (UAE) matrices, defined via three-way co- activation. These higher-order analogues of connectivity matrices involve edge relationships and have a low- rank structure not captured by standard approaches. They also lead to a new concept of edge communities (1- simplexes) that share a triangle (2-simplexes), or maximal edge communities (MEC). In Aim 2, estimated health-associated connectivity patterns in penalized regression models also incorporate higher-order simplicial structures—as predictors and regularizers. These model path structure by viewing node-pairs as boundaries of paths, and modeling their effective resistance (ER), which quantifies network-wide robustness of communication among nodes. Aim 2 leverages the UAE matrix to define a “lifted graph”, and the corresponding lifted-graph Laplacian is used for penalized regression on edges. These models encompass kernel-based methods that involve subject similarities based on simplicial structures. Aim 3 considers matrix- on-scalar regression models to estimate community-level associations between scalar predictors and adjacency-matrix responses. Rather than regressing based on prescribed mesoscale structure associations this form of model is extended to higher-order adjacencies structures, including MECs and other SCx structures. Aim 4 explores the recent concept of persistent Laplacians. This new operator relates the properties of two simplicial complexes when one is embedded in another. This allows analysis of a population of networks/simplices, which do not necessarily share all edges or triangles, by relating them to a common “core” SCx. Participant-wise discrepancies from this core, using the SCx algebra framework, leads to a new type of analysis. Successful completion of the proposed research will provide urgently needed extensions to current analytical methods with new models and software tools aimed at understanding common neurobiological disorders.

NIH Research Projects · FY 2026 · 2026-04

Oxidative protein folding in bacteria PROJECT SUMMARY Oxidative protein folding generates covalent linkages between the sulfur atoms of two cysteine residues, known as disulfide bonds (DSBs). DSBs play a crucial role in the three-dimensional structure and biological function of exported proteins in prokaryotes and eukaryotes. Since the discovery of the first DSB catalyst in the bacterium Escherichia coli, bacteria have been proven to be a powerful model system for understanding the molecular mechanisms that drive DSB formation. Our research aims to address three key questions in the field: 1) What principles enable productive thiol oxidation in essential proteins when canonical DSB-forming enzymes are inactive? 2) What proteins depend on DSBs in complex cell envelopes? and 3) Do distinct thiol-disulfide oxidoreductases exhibit substrate specificity? My lab employs a multidisciplinary approach to dissect the fundamental mechanisms and consequences of DSB formation in vivo using bacteria. As model systems for our basic research program, we employ two model organisms, the Gram-negative bacterium E. coli and the acid-fast bacterium Mycobacterium smegmatis, because (1) they each express one of the two divergent pathways known for DSB catalysis in prokaryotes, (2) they are genetically tractable with a plethora of tools developed over the past three decades, and (3) they have intricate cell envelopes thus many more proteins necessitate oxidative protein folding. The proposed research aims to leverage bacterial genetics, chemical genetics and biochemistry to advance the understanding of DSB formation in bacteria and reveal novel oxidation mechanisms assisting folding with canonical DSB-forming system, the contributions of DSBs in organisms with the alternative system and the determinants of substrate recognition and specificity among different classes of oxidoreductases. Ultimately, our work will inform how DSB forming pathways can be targeted for antibacterial innovation to combat diverse clinically relevant infections and harnessed to engineer proteins with enhanced stability. First, we will elucidate the mechanism of oxidation in essential proteins harboring DsbAB systems. Preliminary Tn- seq screens have already identified synthetic lethal interactions of a dsbA mutant, which represent candidate genes that are likely involved. We will study these candidate genes through genetic and biochemical approaches. Second, we will characterize the DSB oxidation and isomerization systems in the mycobacterial cell envelope using genetic and biochemical approaches. We recently found that DSB formation is crucial for the folding of multiple essential proteins in mycobacteria. We aim to understand DSB formation in two different cell envelope architectures to dissect substrate preference between the two classes of DsbA proteins capable of recognizing hundreds of substrates, through cysteine derivatization and enrichment proteomics. Collectively, our basic research will provide fundamental insights into novel oxidative reactions, the exported proteins that depend on DSBs, and the substrate specificity among thiol-disulfide oxidoreductases.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Microorganisms are frequently challenged by conditions that are suboptimal for growth and reproduction. To persist in such environments, microbes engage in dormancy, a strategy whereby individuals enter a reversible state of reduced metabolic activity. Traditionally, dormancy has been viewed as an adaptation to cope with harsh and fluctuating abiotic conditions, such as resource limitation, extreme temperature, or exposure to antimicrobial agents. However, growing evidence suggests that dormancy plays an important role in modifying host-virus interactions. Although dormancy can provide protection from infection, our findings demonstrate that viruses have evolved strategies to circumvent this metabolic defense. For example, certain viruses hijack the dormancy program of spore-forming bacteria (Bacillus and Clostridia) with the aid of auxiliary genes acquired from their hosts. During this process, viral genomes become entrapped within bacterial spores where they are shielded from the extracellular environment. Upon germination, these non-integrated viruses resume their lytic cycle and exploit the host for replication. Using Bacillus subtilis as a model system, this proposal aims to advance our understanding of host-virus coevolution within the framework of dormancy theory. First, we will test competing models of entrapment using CRISPR gene editing of viruses and quantitative single-cell assays to evaluate the role of auxiliary sporulation genes in lytic suppression and chromosomal segregation. Second, we will investigate the developmental consequences of entrapment based on host-virus expression profiles through single-cell transcriptomics and AI-informed morphological analyses. Third, we will track the fate of infected spores (“virospores”) using a microfluidic platform to quantify the dynamics and consequences of entrapment for host and virus fitness. To synthesize, we will integrate experimental data with mechanistic models to resolve conflicts about virus infection and dormancy decision-making in uncertain environments. The proposed work will advance host-pathogen theory and inform clinical applications, including phage therapies for chronic infections caused by spore-forming bacteria and other microorganisms with quiescent life stages.

NIH Research Projects · FY 2026 · 2026-02

Abstract/Summary: The Fernández Lab seeks to understand how environmental cues and internal physiological states are processed by the nervous system to drive behavior. Our current research focuses on the circadian clock, which allows organisms to maintain internal temporal order and anticipate daily changes in their environment. While the molecular mechanisms underlying the circadian clock are well characterized, the processes by which clock neurons develop and establish connections with each other and with downstream pathways remain poorly understood. Drosophila is an excellent model due to the conservation of the molecular circadian clock and the presence of network motifs resembling those in mammals. Emerging evidence from both mammalian and invertebrate models suggest that core circadian clock genes, some of which are expressed prior to the initiation of the molecular clock, play critical in neuronal development and physiology beyond their established functions in molecular timekeeping mechanism. We recently showed that this is the case for the Drosophila clock gene cycle. When downregulated exclusively during development, cycle leads to altered clock neuron morphology and the loss of adult behavioral rhythms. Our ongoing and future research is aimed at understanding how clock neurons establish their connectivity patterns during development to form a synchronized network of neurons that drives rhythmicity, from gene expression to complex behaviors. Specifically, we will examine the developmental roles of the clock genes cycle and Clock in shaping the connectivity of the main circadian pacemaker neurons and other early-born clock neurons. Notably, these genes are expressed in clock neurons before molecular oscillations can be detected, and we will assess how their downregulation impacts gene expression pathways that influence early-developmental neuronal morphology and connectivity. Additionally, we will examine how various groups of clock neurons respond to key synchronizing signals throughout development, as well as to stimulation of other clock neuron classes. Our recent findings indicate that female circadian rhythms are more resilient to loss of synchronizing factors and suggest that the relative hierarchy of circadian oscillators is sexually dimorphic. We aim to determine the extent to which sexual dimorphism in circadian timekeeping emerges during development and the contributions of sex determination genes in establishing sex differences. Our research program aims to understand how clock neurons develop, communicate, and form precise connections to generate a functional network that drives timekeeping. Our results will contribute to a broader understanding of the mechanisms underlying neuronal development and behavioral plasticity.

NIH Research Projects · FY 2026 · 2026-02

Summary In this proposed research, we aim to develop AI-driven virtual screening pipelines to screen billion-scale compound libraries and predict blood-brain barrier (BBB) permeability, which are critical steps for identifying drug-like lead compounds against AD/ADRD targets, as the current computational methods do not adequately address these crucial needs. First, we will incorporate small molecule foundation models, Bayesian ensemble learning, and active learning to accelerate the docking-based virtual screening pipeline, enabling efficient virtual screening of billion-scale compound libraries. Second, we will incorporate Affinity Selection Mass Spectrometry (ASMS)-based screening and advanced AI/Computational tools to enable similarity-based virtual screening (SBVS) for AD/ADRD targets when their ligand-bound structures are not known. Finally, we will construct a large- scale ΔGSolv dataset and take advantage of the dataset to develop AI/ML models that predict Kp,uu and Pgp ER, providing a more nuanced understanding of brain penetration for small-molecule brain penetrant drugs.

NIH Research Projects · FY 2025 · 2025-09

Project Summary In the United States, there is currently an unprecedented surge in depression rates, particularly affecting low-income women, and especially those who are parents. In part, this surge in depression is due to parenting stress, contextual stressors, and financial strain, and the cumulative impact of these stressful experiences. However, there is a lack of research on maternal depression over long periods of time in low-income samples, which is important because findings from short-term studies of largely higher-income community samples may not extend to other populations. To address this gap, we leverage an existing longitudinal experimental design to prospectively identify variation in trajectories of maternal depression across 25 years in relation to sociodemographic characteristics, child developmental periods, and contextual factors. To capture women’s multiple experiences of stress, we will examine profiles based on contextual risk factors (e.g., neighborhood conditions, social stress, financial strain) and protective factors (e.g., support from family and friends, individual identity, housing stability, neighborhood support) and how they relate to depression trajectories. We will also evaluate whether a family-centered intervention, the Family Check-Up (FCU), might buffer severity in trajectories of depressive symptoms. We will conduct this investigation using the Early Steps Multisite (ESM) Study, a sample of 731 low-income families assessed 13 times from child ages 2 to 19. Families were randomly assigned to the FCU or a control group at baseline. Maternal depression, child/family functioning, and contextual factors have been prospectively assessed for nearly two decades. Families were assessed nearly annually for 8 years post-baseline, and intervention families were offered the FCU. Additional family assessments occurred at 12-, 14-, and 17-year follow-ups (81% retention), and twice in 2021 (18- and 18.5-year follow-ups). For the current project, we plan to re-contact ESM mothers (N = 609; current Mage = 46.90; SD = 6.21) to complete a survey and clinical interview to assess depressive symptoms and disorders, current contextual experiences, and mental/physical health comorbidities. Our overall objective is to extend understanding of long-term patterns of maternal depression in low-income families to inform prevention and intervention approaches that are responsive to various contexts. By identifying social and contextual mechanisms that predict persistence or resilience in maternal depression, we aim to pinpoint contexts in which the FCU might effectively attenuate the risk of enduring maternal depression.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Chronic kidney disease (CKD) is a leading cause of death worldwide. While traditionally associated with older age, hypertension, diabetes and obesity, a new CKD has emerged characterized by occupational origins and a nontraditional, multifactorial etiology. This CKD of nontraditional causes (CKDnt) already accounts for nearly 26 million cases of CKD worldwide. Despite often being attributed to concomitant factors like toxin, disease, and other environmental exposures, evidence supports that CKDnt is associated with work-related heat stress (WRHS) and the development of hyperthermia- (i.e., increased core (internal) body temperature). Hyperthermia exposure can cause clinical or subclinical acute kidney injury (AKI). Clinical AKI or repeated episodes of subclinical AKI (sAKI), defined as increases in AKI biomarkers, leads to a sustained fall in kidney function. Widespread acceptance of this hyperthermia-mediated etiological pathway is complicated by three factors. First, studies have largely been conducted in tropical occupational locations with limited seasonal temperature variation and in workforces mostly free of traditional CKD risk factors. Second, while clinical AKI is a likely cause of CKDnt, the role of sAKI is less certain. Third, hyperthermia appears to be common in workers at risk of CKDnt, but the role of repeated hyperthermia exposures is not well understood. Thus, the long-term goal of this research is to identify solutions that safeguard kidney health during WRHS. The objective of the proposed project is to identify the contribution of hyperthermia exposure to sAKI and CKDnt risk in a climate region with seasonal WRHS and in workers with a relatively high prevalence of traditional CKD risk factors. The central hypothesis is that hyperthermia exposure is a primary driver leading to increased sAKI risk and sustained declines in kidney function among outdoor workers. The central hypothesis will be tested by pursuing three independent specific aims in a project that will employ a longitudinal observational design in which markers of WRHS, hyperthermia exposure, sAKI risk, kidney function, and urinary metabolomic changes will be measured in the winter and late summer months in construction workers at high risk of WRHS associated AKI in the U.S. Aim 1 will identify the role of hyperthermia exposure on cross-shift sAKI risk. Aim 2 will characterize longitudinal changes in kidney function over the U.S. summer. Aim 3 will determine changes in the urinary metabolome in outdoor workers experiencing increased cross-shift sAKI risk. The proposed research is innovative because it will improve the hyperthermia-mediated sAKI-to-CKDnt model by including more varied climatic regions and CKD risk factors. This contribution is significant because the obtained information will provide strong support for a universal hyperthermia-mediated sAKI-to-CKDnt etiology and pave the way for therapies targeting metabolic pathways to prevent hyperthermia-mediated sAKI and, ultimately, CKDnt. It is expected that this contribution will provide the foundation for protecting outdoor workers against sAKI and CKDnt against a backdrop of hot weather.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Gene replacement therapy for inherited retinal degenerations has improved visual function in animal models, which has built momentum to curing blindness in humans. Optimal therapy is the return of normal visual function, however, current clinical trials face challenges associated with variability and durability of recovery due to the lack of rigorous mechanistic understanding of the retinal circuit's reaction to the therapies and any potential hinderance to full recovery. To restore vision, it is essential to understand how surviving retinal neurons modify synaptic connections upon vision restoration treatment and how retinal plasticity can be leveraged to improve visual function. Our long-term goal is to elucidate fundamental mechanisms that enable the retina to establish functional connections following gene therapy. The objectives of the proposed work are to determine underlying mechanisms of functional recovery at cellular and circuit resolution using a mouse model of achromatopsia, which restores selective loss of cone-mediated function after gene therapy. In Aim 1, we will determine the recovery of spatial and temporal processing in ON and OFF pathways after gene therapy. We will measure spatio-temporal receptive fields of specific ganglion cell types. In Aim 2, we will determine the contribution of synaptic remodeling and transmitter release homeostasis to the structure and function of cone bipolar cells following gene therapy. Achieving robust and sustained therapies require understanding of how gene therapy restores rewiring and neurotransmitter release from first- and second-order synapses. Imaging and electrophysiology will allow us to determine the wiring patterns of outer and inner retina, and how cones and bipolar cell release rates potentially adapt to changes in inputs to reach homeostasis. The approach is innovative for a new perspective on restoring vision in the context of the retinal plasticity investigating the effects of gene therapy on connectivity patterns and functional properties at the single-cell level of the retinal circuit. The results will be significant for (1) revealing whether retinal plasticity is constructive toward restoring visual function, (2) determining mechanisms that allow the remaining retinal neurons to re-establish functional connections with newly rescued cones, and (3) providing knowledge essential for maximizing function after photoreceptor recovery.

NIH Research Projects · FY 2025 · 2025-09

Abstract/Project Summary The fluorescence lifetime of a dye can inform about cellular signaling, protein-protein interactions, and membrane microenvironments with fluorescence lifetime imaging microscopy (FLIM) and time-resolved flow cytometry (TR- Flow Cytometry). These techniques primarily use changes in the fluorescence lifetime from Föster-Resonance Energy Transfer (FRET) dye pairs to convey biological information occurring within a 10-nm distance. In addition, FLIM is often used to circumvent biological autofluorescence by producing an image based on the differences in the fluorescence lifetimes of a dye. However, the fluorescence lifetimes of archetypal dyes are short (< 3 ns), and their minimal lifetime changes upon biological stimuli cannot be resolved with these instruments, limiting the extent and precision of biological interrogation during their operation. The goal of our program is to synthesize, characterize, and implement a palette of target-specific organic dyes with biologically-responsive long-lived fluorescence lifetimes, primarily by incorporating thermally activated delayed fluorescence (TADF) photophysics. Of biological relevance is that these organic dyes can yield two long-lived fluorescence lifetimes that are mechanoresponsive, thermoresponsive, and sensitive to voltage. Therefore, we hypothesize that concomitant changes in the fluorescence lifetime of our optical reporting dye technology will be easily resolved with FLIM and TR-Flow Cytometry and inform us of viscosity, polarity, and voltage changes associated with biological function. In addition, this TADF dye technology can potentially extend the Föster resonance distances of the FRET dye pairs, opening new frontiers for interrogating biomolecules separated by distances > 10 nm. Of relevance is sensing cholesterol, as its levels impact membrane viscosity. Our preliminary data demonstrate that a TADF dye can sense cholesterol levels in liposomes, as its fluorescence lifetime increased from 9.4 ns to 13 ns when cholesterol levels were increased from 0% to 20%. Contrarily, a constant fluorescence lifetime of ~ 2.6 ns was obtained for DI-8-ANEPPS, a cholesterol-sensing dye. Notably, a long-lived fluorescence lifetime of 711 ns was detected for the TADF dye in giant multilamellar vesicles (GMLVs) that was not clearly detected in liposomes. These results are significant, as these lifetime increments are easily resolved with fluorescence lifetime measurements and are a goal for dye synthesis. A plausible explanation is that the lifetime differences between GMLVs and liposomes are due to membrane curvature differences, confirming that TADF dyes can be used to inform membrane morphology. Building on these findings, we are synthesizing a palette of TADF dyes that fully intercalate into membranes, target the mitochondria and important organelles, and serve as bioconjugates for biological interrogation with FLIM and TR-Flow Cytometry. To achieve this objective, our program combines previous success in organic dye synthesis, photophysical characterization of organic TADF dyes, and biophysical interrogation in artificial and living systems. Our program can have applications in medical diagnostics, cellular trafficking, and other biological research beyond the immediate scope of this initial work. 1

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Sexual strangulation (often called “sexual choking” by those who engage in it, even though it is technically a form of neck compression or strangulation) has become highly prevalent among young adults, is usually described as consensual, and disproportionately impacts females. Our survey of randomly sampled college students showed that 58% of females had ever been choked/strangled during sex. A series of pilot studies have begun unraveling the effects of this emerging and risky sexual behavior, where females with frequent exposure to sexual choking/strangulation showed elevations in brain injury blood biomarkers, increases in mental health symptoms such as feeling depressed and anxious, alterations in brain morphology, and changes in neuronal connectivity. While relentless efforts through epidemiological, qualitative, and experimental studies have greatly advanced the scientific knowledge of this sexual behavior, the current understanding of neurological consequences has been limited by cross-sectional designs and small sample sizes. Therefore, the proposed prospective cohort study moves the field forward by examining acute and chronic neurological effects of sexual choking/strangulation while a structured interview aim will help us to better understand why people engage in this risky sexual behavior, despite its risks. The proposed study is guided by the following 3 specific aims. (1) To identify the acute neurological effects of sexual choking/strangulation through the response profile of multimodal neuroimaging, blood biomarkers, and cognitive assessments. (2) To determine the associations between cumulative exposure to sexual strangulation and neural cellular, physiological, and functional integrities. (3) To characterize young adults’ experiences with sexual choking/strangulation. The longitudinal design will allow us to examine acute effects of this sexual practice (Aim 1), observe the cumulative impacts of multiple choking/strangulation events (Aim 2), and establish temporality between sexual choking/strangulation and neurological outcomes. Our rigorous interview will help characterize this sexual behavior (Aim 3), which is vital for developing guidelines and educational intervention. The proposed study will not only characterize the impact of sexual choking/strangulation on brain health but also lay the foundation for the development of empirically supported sexual health educational guidelines to mitigate neurodegenerative conditions.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Over the past several decades, advancements in automated technologies for liquid handling and monitoring have significantly accelerated research across various fields of chemistry, biology, and medicine. These innovations have facilitated breakthroughs in numerous applications, including sequencing library preparation, clinical diagnostics, large-scale compound screening, and high-throughput chemical reactions. Usually, these biological, biochemical, and physical reactions/processes occurring within tiny droplets (with a volume of picolitre) may exhibit heterogeneous, complex, and dynamic characteristics, requiring high-throughput and high-precision handling, real-time mentoring, and dynamic feedback control of massive liquids. Current liquid handling methods using robotics, micro-droplets, pneumatic valves, electrowetting, acoustics, hydrodynamics, or magnetics have achieved fluid processing, but some may face significant issues including cross-contamination, low throughput, limited precision, and reproducibility. Moreover, current automated liquid handling technologies lack real-time monitoring and dynamic feedback control, limiting their ability to handle various reagents and manipulate complex conditions. To address these challenges, we propose an ‘Intelligent Droplet Acoustofluidics’ platform. Our recent invention of ‘Digital Acoustofluidics’ integrates acoustics and microfluidics for manipulating droplets in oil, showing promising potential to overcome the drawbacks of the existing liquid-handling technologies. Additionally, our engineering advances in ‘Intelligent Acoustofluidics’ leverage artificial intelligence (AI) for dynamic regulation of acoustofluidic actuators via an imaging-based closed-loop feedback control system. By combining two unique systems together, we expect the platform can achieve on-demand generation of droplets with controlled volumes, transportation of multiple droplets in parallel, fusion and mixing of multiple droplets, real- time monitoring of each droplet, and automated operation of liquid-liquid interaction using an AI-guided closed- loop controller. After optimizing the proposed platform, we will validate the performance of the proposed platform across two distinct biomedical applications: protein crystal chemistry and high-throughput drug screening to address the limitations that have hindered in using of traditional liquid-handling tools.

NIH Research Projects · FY 2025 · 2025-09

The straightforward laboratory preparation of structural motifs commonly found in therapeutic agents is a critical driver for the design and development of new and more efficient chemical reactions. This is especially true in the pharmaceutical industry where efforts to increase the 3-dimensional complexity of pharmaceutical lead candidates has resulted in substantial challenges related to their efficient and cost-effective preparation. While our capacity to make complex molecules has become greatly developed, the preparation of stereochemically complex targets in useful quantities remains very difficult. Accordingly, there is a critical need for the design and development of new and efficient stereoselective reactions with which to address this challenge. The long-term objective of our research laboratory is to introduce a general, modular, and operationally trivial toolbox of effective stereoselective reactions. Within this remit, we have leveraged both Lewis base and transition metal catalysis as sources of unique and enabling reactivity but have also embraced cooperative catalysis as a general blueprint for reaction design. Cooperativity emulates mechanisms commonly encountered in enzymes, exploiting synergy between simultaneous yet complementary catalysis control bond formation. Our rationale is that, by making two catalysts work together, greater control over reactivity and stereoselectivity could be possible than when using either catalyst in isolation. This will allow us to design entirely new chemical reactions that work by entirely new reaction mechanisms. This promises much for the design of new, efficient, and operationally trivial catalytic stereoselective reactions with which to expedite the design, development, and manufacture of medicines to manage and treat diseases. Based on exciting preliminary data we have obtained in enantioselective Lewis base catalysis and Lewis base/transition metal cooperativity — the research described in this proposal will provide the scientific community with straightforward, reliable and flexible methods for chemical synthesis. This work will significantly impact human health and medicine by establishing routine synthetic protocols for the preparation of valuable fluorinated molecular scaffolds, which will contribute to the design and development of new clinical agents.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY The transition of bacteria from a free-living state to a sessile, surface-attached growth mode often leads to formation of multicellular biofilms, profoundly altering cellular physiology and increasing antimicrobial tolerance. Attachment and biofilm formation is regulated in many systems by the pivotal cytoplasmic second messenger cyclic diguanylate monophosphate (c-di-GMP), with high levels commonly promoting adherence and low levels favoring planktonic growth. Recent work has identified a remarkable regulatory circuit that controls intracellular c-di-GMP levels through the activity of excreted metabolites called pterins. Diguanylate cyclases (DGCs) synthesize c-di-GMP from GTP, and phosphodiesterases (PDEs) degrade the signal, with certain enzymes that have both activities, the balance of which is often under environmental control. In Agrobacterium tumefaciens, a plant pathogenic Proteobacteria, the DcpA protein is a dual function DGC-PDE, that requires excreted pterins to maintain a PDE-dominant activity in planktonic culture. Most forms of life synthesize pterins from a branch of the folate biosynthesis pathway and in bacteria their excretion is recognized but poorly understood. In A. tumefaciens pterins are released into the periplasm, where they interact with PruR, a pterin binding protein. Pterin-associated PruR interacts with the DcpA periplasmic domain, and biases the protein to a PDE-dominant state. Specific reduced pterins generated by pteridine reductases in the cytoplasm, are preferred ligands for PruR, and mutants that do not produce these reduced pterins switch DcpA from PDE-dominant, to a strong DGC activity, stimulating attachment and biofilm formation. PruR-DcpA regulatory circuits are broadly, but non- uniformly conserved among the Proteobacteria and comparative structural analysis predicts that most of the PruR-type proteins will bind pterin-type molecules. The proposed study is designed to test and expand the current PruR-DcpA model in A. tumefaciens, as a prototype for related bacterial systems, using a combination of molecular genetics, biochemistry, synthetic organic chemistry, genomics and bioinformatics. The research plan will perform in depth interrogation of pterin-PruR binding and determine how this in turn regulates DcpA activity. The production and excretion of pterins as a branch of the folate pathway, and to what extent this impacts PruR-DcpA regulation, will be studied using antibiotics, targeted genetic manipulation, and an unbiased mutant screen. The phylogenetic distribution of PruR-DcpA-type systems will be extensively investigated, elucidating additional genetic relationships, patterns of conservation, and paths of evolution. Expanding beyond the A. tumefaciens system, a genetically tractable opportunistic human pathogen, Aeromonas veronii with four PruR-DcpA modules, will be dissected using heterologous expression, genetics, and biochemical approaches to disentangle their regulatory roles. This work will provide numerous insights into this newly discovered, widely relevant mechanism controlling biofilm formation and likely other yet to be identified processes, with the potential to lead to new therapeutic targets.

NIH Research Projects · FY 2026 · 2025-09

Youths and young adults with attention-deficit/hyperactivity disorder (ADHD) are at increased risk for substance use disorders (SUD) and other SU-related morbidity and mortality. Clinical guidelines recommend pharmacotherapy as part of multimodal treatment, but the existing research estimating ADHD medication effects on SU problems is mixed and inconclusive. These inconsistencies are made more complex by several significant knowledge gaps that directly impair the clinical care of young people with ADHD. First, little is known about medication utilization and medication effects on SU problems among young people at high risk for such outcomes in the U.S., especially those enrolled in Medicaid. Plus, we have limited understanding about medication effects among key at-risk subgroups, including those who initiate pharmacotherapy after childhood or have co-occurring SUD. Second, we need a better understanding of the use and effects of different pharmacotherapies, including nonstimulant medications such as atomoxetine and alpha agonists, on SU problems. Possible dose-response effects of stimulants on SU problems are also poorly understood. Finally, there is no consensus on the effects of discontinuing long-term ADHD pharmacotherapy on SU problems. There is, thus, a critical need to characterize the patterns and consequences of initiating and discontinuing ADHD pharmacotherapy, particularly among understudied populations, such as those enrolled in Medicaid. The overall objectives of this application are to (a) characterize the treatment of ADHD in youths and young adults enrolled in Medicaid and (b) estimate the effects of different medications and dosages on serious SU problems (i.e., SUD and fatal and nonfatal overdose). We will pursue these objectives by analyzing data from Medicaid enrollees nationwide that includes 3.3 million individuals aged 9-29 years old with a diagnosis of ADHD from 2016-2020. We will characterize medication utilization heterogeneity and assess medication effects using several designs that reduce biases in observational studies, including (a) within-individual (or ‘self-controlled’) designs that implicitly rule out all time-stable confounding and account for time-varying factors via statistical controls and (b) target trial emulations. The rationale for the proposal is that analyzing national, real-world healthcare data with these advanced methods can more precisely characterize the treatment of young people with ADHD and the effects of different medications on serious SU problems. We will examine all young people with ADHD on Medicaid, as well as subgroups whose age or cooccurring SUD may impact treatment outcomes through three aims. First, we will characterize real-world patterns of ADHD medication treatment. Second, we will estimate the effects of initiating different ADHD medications (and doses) on SU- related morbidity and mortality. Third, we will estimate the effects of discontinuing long-term ADHD medication treatment on these outcomes. We expect this work will inform personalized clinical decision-making and evidence-based treatment policies, especially via our “user-centered epidemiology approach.”

NIH Research Projects · FY 2025 · 2025-09

Project Summary Vaginal dryness is a condition that affects millions of women, especially as they age but also as a side effect of many commonly prescribed drugs. Frequently painful, vaginal dryness affects quality of life and increases risk of other disorders including infections. Prescribed therapies for premenopausal vaginal dryness are limited. Many important drug classes including many antipsychotics, antidepressants, antihistamines and antibiotics cause vaginal dryness as well as dry mouth and eye, and these contribute substantially to patient non-compliance. The subject of vaginal dryness has seen limited study, in part for want of animal models. Mice are a valuable first-line animal model accompanied by an extraordinary range of transgenic variants. Mice have not been used for studies of vaginal function partly because of the challenge of reliably measuring small volumes of vaginal secretions. But it is also frequently assumed that the mouse reproductive system isn’t close enough to the human to be relevant. We have developed a novel method to quantify vaginal secretion in small animals. This proposal tests whether mice may serve as a research model for a series of conditions that impact vaginal function in humans. The assay itself is rapid, minimally invasive and readily repeatable. In mice this assay is sufficiently sensitive to measure both increases and decreases in vaginal secretion. Preliminary data indicates that murine vaginal secretion can be stimulated by a volatile chemical messenger. There are many conditions that contribute to vaginal dryness, from inflammation (e.g. Sjogrens) to polypharmacy. The proposed project tests a panel of these to determine the relevance of the mouse as a model of human vaginal function. It is tempting to dismiss the mouse as a model for studies of vaginal function in humans. Given the gravity of women’s health issues and the limited experimental tools available, we can ill afford to dismiss the mouse a priori without investigation. The experiments proposed here will provide answers to this question and even a negative answer will be valuable for researchers. Positive results would set the stage for further research studies that can contribute to women’s health.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY/ABSTRACT While extinction learning can reduce excessive fear characteristic of anxiety-based disorders like post-traumatic stress disorder and generalized anxiety disorder, behavioral inhibition achieved through extinction is highly susceptible to relapse. This is because extinction functions to create a new inhibitory memory while leaving the original fear memory largely unaltered and intact. We have recently identified and characterized behaviorally a novel approach to behavioral reduction that seems to circumvent the behavioral relapse common to extinction. In this procedure, weak versions of the fear-provoking unconditional stimulus are presented to alter the representation of that fearful stimulus to ultimately weaken fear responding to cues previously associated with it. The present proposal aims to expand on our understanding of this phenomenon and apply it to novel situations to reduce behavioral relapse following fear-reducing procedures. Our overarching hypothesis is that unconditional stimulus deflation will be distinct from extinction at the behavioral, circuit, cellular, and molecular levels and will be tested in three complementary but independent aims. In Aim 1, we will first directly compare UCS deflation to extinction in the three most commonly-studied relapse paradigms: renewal, spontaneous recovery, and reinstatement with the prediction that UCS deflation, but not extinction, will reduce all three types of relapse. Then, we will test the requirement for neural activity in the IL BLA pathway during UCS deflation in creating these long-lasting reductions in fear responding using an optogenetic approach. In Aim 2, we will examine the degree to which extinction and deflation activate the original memory ensemble using a robust activity marker (RAM) approach. Here, our prediction is that UCS deflation will preferentially activate the memory ensemble tagged during acquisition, with less activation of this ensemble in extinction learning. We will then quantify the degree to which the original training ensemble is activated following renewal testing. Here, we hypothesize more overlap between the neural ensemble in renewal following extinction than following UCS deflation. Finally, we will test the prediction that the ubiquitin proteasome system will have a unique role in UCS deflation but not extinction. First, we will examine changes in nuclear and synaptic K48 polyubiquitin tagging following UCS deflation or extinction with the prediction that UCS deflation will produce changes in the synapse indicative of memory retrieval and updating whereas extinction will produce changes in the nucleus indicative of new memory formation. We will then test the hypothesis that blocking proteolytic activity will impair UCS deflation but not extinction using an intracranial approach. These studies will be the first to examine how UCS deflation functions to reduce fear across multiple levels of analysis (behavioral, circuit, cellular, and molecular). Further, they will provide detailed mechanistic insight and identify specific targets through which UCS deflation can weaken the original learning to persistently reduce fear and render this reduction less susceptible to relapse.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Rates of the human disease vibriosis caused by Vibrio bacterial infections are steadily increasing world- wide due to rising ocean temperatures, surges in Vibrio abundance, and broader bacterial distribution in aquatic ecosystems. Due to multi-drug resistance, there is an urgent need to develop alternative and innovative strategies beyond standard antibiotics for treating Vibrio infections. A major pathway that drives pathogenesis in Vibrios is quorum sensing (QS): bacterial cell-cell chemical communication. QS signaling culminates in the production of a TetR-type master transcription factor collectively called the SmcR family, which regulates genes required for infection of host organisms. Thus, it is postulated that SmcR-type proteins are optimal targets for drug development to inhibit QS and treat vibriosis. Previous work by the van Kessel group showed that thiophenesulfonamide compounds such as PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H- pyrazole) are potent and specific inhibitors that bind in the ligand binding pocket of SmcR-type proteins in multiple Vibrio pathogens. The long-term goal of this work is to develop PTSP and its derivatives into potent anti-QS molecules that treat vibriosis infections in humans. The van Kessel and Paczkowski groups collaborated on structure-function analyses to identify PTSP-interacting residues in the ligand binding pocket required for inhibition of SmcR activity in vivo. The results show that thiophenesulfonamides specifically bind SmcR proteins in multiple Vibrios, alter the conformation of the DNA binding domain, and promote protein degradation, thereby suppressing downstream gene expression. Collectively, these substantial preliminary data implicate ligand binding as a mediator of SmcR protein stability to govern virulence gene expression in Vibrio pathogens. However, the ligand binding pocket of SmcR proteins differ widely among Vibrios and the native ligand for the SmcR family of proteins is unknown. Thus, optimal inhibitor design for multiple Vibrio species will require a thorough understanding of SmcR-ligand interactions. The central hypothesis is that the active conformation of SmcR is driven by native ligand binding. The objective of this proposal is to use computational, biochemical, genetic, and biophysical assays to determine the interactions that drive SmcR- ligand specificity via three aims: 1) perform structure-activity relationship analyses of a thiophenesulfonamide library against the SmcR family, 2) determine the structural basis of ligand-induced conformational changes in SmcR proteins, and 3) identify the native ligand(s) of SmcR. These experiments will determine the molecular changes to Vibrio SmcR proteins induced by ligand binding, which will facilitate inhibitor optimization to enhance efficacy, stability, and pharmacokinetic properties for drug design treatments for vibriosis disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The exposome is defined as the totality of exposures with which the public comes in contact, including toxic chemicals. Exposures to these chemicals represents a huge burden on human health and diseases. It is difficult to perform comprehensive safety assessment of all novel chemicals due to limited time and funds. However, with the vast amount of biological data related to thousands of exposures and their molecular targets, we hypothesize computational methods can be developed to accurately predict the molecular actions and targets of new chemicals. In this proposal, we propose to implement and apply a novel matrix completion algorithm named Coupled Matrix/Tensor-Matrix Completion (CM/TMC) and Coupled Matrix/Tensor-Tensor Completion (CM/TTC) to predict the molecular targets and target tissues of environmental chemical exposures at a large scale. The study proposed will be accomplished through the following specific aims: 1) Apply and optimize the CM/TMC algorithm for exposure-related datasets, comparing results to alternative methods, 2) Optimize the CM/TMC method for exposure target tissue prediction, and 3) develop CM/TTC method on exposure-target predictions, perform experimental validations, and establish a web portal for exposure-target prediction. This study poses the first matrix completion-based method on exposure molecular target predictions and target tissue predictions. The primary goal of the mentored (K99) phase of the award is to provide the candidate with additional training in data science and toxicology for him to acquire scientific independence and successfully accomplish his career objectives. The K99 phase will be conducted at the University of Michigan (UM), under the mentorship of Drs. Maureen Sartor, Justin Colacino, Kayvan Najarian, and Mario Medvedovic, who are experts in the respective fields. An interdisciplinary team of advisors will assist the candidate in his research and career development. After the completion of the K99 phase, the candidate will be well prepared to be an independent investigator.

NIH Research Projects · FY 2025 · 2025-07

Safe sanitation is one of United States’ greatest public health achievements. Yet, regions remain where sanitation infrastructure is lacking that causes an attributable burden of disease. In the United States, this sanitation deficit includes combined sewer systems which are present in 700 municipalities and discharge 850 billion gallons of untreated wastewater to waterways annually. These overflows result in a wide range of exposures including during aquatic recreation, drinking water contamination, food contamination via agricultural irrigation, pathogen accumulation in fish and shellfish, and via mechanical vectors such as flies. Yet, the health impact of sanitation infrastructure upgrades aimed at reducing combined sewer overflows is largely uncharacterized. Enteric pathogens – which may cause diarrheal disease – and antimicrobial resistant organisms may be shed in human feces and are present in high concentrations in sewage. Recognizing the link between sanitation and public health, the city of Indianapolis is investing $3 billion in new sewerage that will nearly eliminate combined sewer overflows. The city experiences up to 90 sewer overflow events annually, which creates the unprecedented opportunity to study the environmental and health impacts of this intervention. Pre- intervention samples will be collected by the study team in 2025, construction will be completed in December 2025, and post-intervention samples will be collected in 2026. The proposed study will examine in detail the results of this sanitation intervention. Assessing the health impacts of city or neighborhood scale infrastructure is complicated by long construction timelines and it is difficult to randomize large infrastructure projects. This study aims to develop a novel framework using high level environmental outcomes to assess city scale sanitation interventions, which could be deployed globally. Surface water samples and flies will be collected and analyzed for fecal indicator bacteria, enteric pathogens, antimicrobial resistance genes, and fecal source tracking markers. The study will quantify the reductions of these targets in an intervention and control area. With this empirical data, quantitative microbial risk assessment (QMRA) will be used to determine the enteric infection risks attributable to aquatic recreation, use of contaminated surface water for agricultural irrigation, and contamination of food via flies. Novel risk assessment methods – that generate ratio measures of effect from QMRA outputs – will be used to evaluate the intervention. If successful, this study will advance our understanding of how to prevent the transmission of enteric diseases in communities reliant on combined sewer systems and combat the dissemination of antimicrobial resistant organisms.

NIH Research Projects · FY 2025 · 2025-07

Abstract The incidence of infants with prenatal opioid exposure (POE) has increased significantly in the last decade. Infants exposed to opioids prenatally are at risk for poor cognitive, behavioral, and neurodevelopmental outcomes later in life. Moreover, a large percentage of infants with POE exhibit a broad variety of neonatal opioid withdrawal symptoms (NOWS) that range from mild to severe. Currently, there is no objective strategy to identify severity and/or guide clinical intervention for NOWS, leading to prolonged hospital stays and delayed treatment. The absence of a unified management strategy is partly due to a lack of quantitative tools to objectively identify NOWS biomarkers in the first days of life. MRI studies linking brain abnormalities with prenatal opioid exposure suggest that cerebral physiology could serve as an early biomarker, but logistical challenges limit its use. Optical techniques like near-infrared spectroscopy (NIRS) offer a non-invasive and cost-effective alternative, but commercial devices are limited in providing a comprehensive profile of cerebral physiology. This project proposes using a novel hybrid device that combines frequency-domain NIRS (FD-NIRS) and diffuse correlation spectroscopy (DCS) to quantify cerebral oxygenation and cerebral blood flow in neonates. While similar in concept and ease of use to standard FDA- approved cerebral oximeters, our approach is more advanced because it can estimate cerebral oxygen metabolism by combining cerebral oxygenation and cerebral blood flow measurements. In this study, we propose to acquire longitudinal FD-NIRS/DCS measurements on infants with POE and controls to determine early differences in cerebral physiology in the first week of life (Aim 1), evaluate their associations with short-term clinical outcomes (Aim 2), and monitor potential changes in cerebral physiology in response to pharmacologic treatment for severe NOWS (Aim 3). No prior study has assessed the potential of bedside measures of cerebral physiology to screen neonates with POE and/or NOWS. Successful completion of our research will offer new insights into the underlying pathophysiology of early brain changes resulting from POE and demonstrate the prognostic value of longitudinal FD-NIRS/DCS measurements for early assessment of NOWS severity in individual neonates. This could potentially improve symptom monitoring and guide personalized treatment, ultimately enhancing the care of newborns with NOWS.

NIH Research Projects · FY 2025 · 2025-07

Project summary/abstract (30 lines max) Substance use disorders (SUDs) remain difficult to treat, but recent studies have shown remarkable, effortless loss of addiction to both opioids and alcohol with invasive deep brain stimulation of the nucleus accumbens. Likewise, disruption of the anterior insula has also led to spontaneous, effortless loss of addiction. While highly effective, deep brain stimulation as a treatment for addiction is out of reach for the general population due to the expense and risk of the brain surgery involved. If similar stimulation could be carried out non-invasively, it could potentially make more effective treatment for a variety of SUDs broadly accessible, with profound individual and public health implications. A new technology called temporal interference (TI) electrical neurostimulation may provide such a means of non-invasive stimulation of deep brain regions, but it has not yet been tested for efficacy in clinical samples. We recently provided a first step by demonstrating that TI can effectively activate the nucleus accumbens as measured by simultaneous fMRI BOLD imaging. With the technique now demonstrated, we propose a preliminary clinical trial to test the effects of 60 minutes of TI stimulation on the nucleus accumbens and anterior insula on nicotine craving and use, both during TI stimulation and for one week following, using ecological momentary assessment methods. Heavy smokers and vape users will be recruited and will abstain from nicotine prior to stimulation. Subjects will be randomized into three groups: nucleus accumbens, anterior insula, and sham control groups. They will receive TI stimulation (or sham) for 60 minutes with self-reported cravings every 10 minutes and each day for one week after. Our existing custom vape device will measure the volume of inhaled vapor to identify changes in actual nicotine use during TI stimulation. All subjects will complete a set of questionnaires assessing their level of nicotine dependence, their comfort during the stimulation experience, their cognitive abilities before and after stimulation, and any changes in emotion and motivation before vs. after the stimulation. We will analyze the results to identify decreases in nicotine craving and/or nicotine vapor inhalation during stimulation of nucleus accumbens vs. sham and anterior insula vs. sham, and directly between nucleus accumbens and anterior insula stimulation. Furthermore, we will identify the duration of such effects by running similar statistical tests on the self-reported craving and nicotine use over the following 7 days. We will also assess safety and tolerability, which were good in our pilot subjects. Positive results in this clinical trial will have profound implications, as the TI technology can be mass produced and used as an “electroceutical”, with equipment potentially similar in size and cost to a common cell phone.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract (30 lines of text) We propose the research and development of a novel, 3D, immersive, virtual reality (VR) exploration tool using a federated collection of datasets from three Common Fund Data Ecosystem (CFDE) efforts: Human BioMolecular Atlas Program1,2 (HuBMAP), Cellular Senescence Network3 (SenNet), and Genotype-Tissue Expression4 (GTEx). The Aim of this proposal is to Enable FAIR5 access to datasets from these three Common Fund programs via an immersive VR interface with embedded 2D/3D visualizations. Using existing, Linked Open Data (LOD) infrastructure built for the Human Reference Atlas (HRA)6–9, we will Federate cell summaries from datasets and extraction sites across three Common Fund programs in the HRA6–9 (Sub-Aim 1), i.e., we will identify datasets that have been registered into HRA 3D Reference Objects (organs) via dedicated tools8, thus giving them a 3D extraction site; then, we will retrieve existing cell summaries for these extraction sites and registered datasets given existing cell-by-gene matrices or proteomics markers (462 datasets as of June 14, 2024). For the related Sub-Aim 2, we will Deploy CFDE datasets in the HRA Organ Gallery in VR, an existing research prototype for a VR application that allows the immersive exploration of this multiscale, multimodal data. Building on our prior research on Data Visualization Literacy, VR, and immersive analytics10–12, we will add 2D and 3D visualization capabilities to the HRA Organ Gallery to visualize cell summaries at the dataset, 3D extraction site, and anatomical structure (atlas) level. All cell summaries will be connected to 3D registration sites inside one of 65 existing 3D reference organs (spatial), annotated with anatomical structure(s) that are aligned to the UBERON13 ontology (biological structure), and associated with clinical (donor) metadata, such as age, sex, race, etc. All work and code will be documented and made available to the community. Additionally, we will update existing and create new training and instructions materials, not only in written form, but also as videos and inside the HRA Organ Gallery in VR application. Further, we are planning extensive demonstrations of this work at CFDE meetings and at various events organized for HuBMAP, SenNet, and GTEx researchers.

NIH Research Projects · FY 2024 · 2025-06

Project Summary/Abstract The advancement of antiretroviral treatment (ART) has significantly reduced the HIV mortality and morbidity by reducing the viral load (VL) to undetectable levels. Recently revised WHO guidelines strongly recommend routine VL testing to monitor ART adherence and minimize failure. To this end, HIV self-testing, a process in which individual who wants to know HIV status collects a specimen, performs a test and interprets the result in private, has become an empowering approach. While nucleic acid testing (NAT) is readily available in centralized laboratories for viral load quantification, its availability in self-testing has not been demonstrated due to sample processing and assay complexity. This project aims to develop a quantitative test on an ultra-compact USB device to detect viral rebound that is simple enough for laypersons to test themselves in the United States. The R61 phase of the project will develop the whole blood-based test that can quantitatively assess the presence of HIV-1 RNA at concentrations as low as 1000 copies/ml, threshold recommended by WHO for determining treatment failure. In aim 1, we will develop a disposable microfluidic chip for streamlined and automated plasma separation and viral RNA extraction from whole blood. In aim 2, we will optimize the HIV-1 RT-LAMP assay and explore the minimum copy number sensitivity. In aim 3, we will integrate the quantitative USB analyzer hardware and develop software for easy and robust operation. In aim 4, we will validate the prototyped test in the BSL-2 lab using HIV-1 plasma samples spiked into whole blood. The R33 phase of the project will assess the test performance, usability and stakeholder needs within the HIV Comprehensive Care Program at Penn State Hershey Medical Center. In aim 5, the performance of the proposed test on HIV-infected patients will be benchmarked with standard laboratory methods. In aim 6, we will evaluate the user’s experience, attitude and perception of HIV VL self-testing. Through innovations in microfluidic chips and the USB analyzer, we anticipate the test would be able to quantify HIV-1 VL as low as 1000 copies/ml directly from 100 µl of finger prick blood. The potential impact of this project is very high. If this proof-of-concept project is successful, it has the potential to significantly enhance the treatment outcomes for individuals of HIV under therapy. The potential transformative capacity warrants the challenges associated with this project.

NIH Research Projects · FY 2025 · 2025-05

Project summary/abstract (30 lines max) A number of clinical disorders such as addiction, OCD, and schizophrenia, as well as neurological disorders including Parkinson’s disease, depend critically on deeper brain regions. Invasive deep brain stimulation of the nucleus accumbens, for example, has been shown to cause rapid, effortless loss of addiction, but these interventions are impractical due to the risks and costs of brain surgery. There is currently no developed non- invasive method of deep brain stimulation -- existing methods such as TMS, tDCS, tACS, and tFUS do not stimulate deep brain regions without also stimulating the overlying cortex, causing unwanted side effects and confounds. Moreover, there is unresolved debate about the causal role of deeper brain regions in cognition, as methods like fMRI show correlations but not causation between brain activity and behavior. This project explores temporal interference (TI) electrical neurostimulation as a potential new technology to test causal hypotheses about deep brain function in human cognition, which may further provide a foundation for treating clinical disorders involving deeper brain regions. TI works by applying two or more high frequency alternating current fields of slightly different frequencies. The fields individually do not stimulate brain activity, but where the fields overlap, there is a pattern of temporal interference which can activate neurons. Using combined TI and fMRI in human subjects, we will characterize the effect of TI on BOLD signals and functional connectivity, and how those vary with different TI frequencies and targeted brain regions (Aim 1). Then we will apply TI to the dorsal anterior cingulate cortex (dACC), to resolve longstanding debates about the causal role of dACC in cognition, especially in conflict monitoring, risk avoidance, and foraging behavior (Aim 2). The results will establish TI as a means of safely and effectively manipulating deep brain activity without activating the overlying cortical regions. This in turn will provide a new method for answering questions about the causal role of various deeper brain regions in cognition as well as a new means of treating clinical disorders involving deeper brain regions.