Virginia Polytechnic Inst And St Univ

NIH Research Projects · FY 2024 · 2024-07

SUMMARY The International Gap Junction Conference (IGJC) is a biannual international meeting that has been alternating between Europe and America since 1987. This meeting is the only one of its kind that gathers scientists and clinicians from all over the world to present and discuss research on connexins, pannexins, innexins, and gap junction proteins in general. The most well-known connexin-mediated function is perhaps in formation of gap junctions within cardiac muscle which provide direct intercellular electrical coupling necessary for each heartbeat to occur. Alterations in connexin regulation and function are understood to cause arrhythmias of sudden cardiac death, and a significant portion of IGJC2024 will be dedicated to normal and pathological mechanisms of connexin regulation encompassing transcription, translation, post-translational modification, channel biophysics/structure, and stability/degradation. In addition to intercellular communication, connexin hemichannels occur on the cell surface, and increasing non-junctional cytosolic functions of connexins are being uncovered. Pannexins share a protomeric structural similarity with connexins but are not considered to form intercellular channels, but rather function intracellularly or as cell-surface channels controlling release of ATP and other metabolites for purinergic signaling. Just as with connexins, but through distinct mechanisms, pannexin function is closely related to heart and vascular biology, with significant roles in heart disease progression and blood pressure maintenance, for example. Pannexins have evolved from innexins, which form gap junctions in invertebrates. There has been a recent resurgence in innexin research using model organisms which is providing further insight as to evolution and mechanisms of pannexin/connexin structure and function. By combining these three dynamic fields of research, the IGJC provides a uniquely diverse and rich intellectual space with a demonstrated history of fostering collaboration to move the field forward and providing support and exposure for early career investigators to share their science. It is our overall Aim to ensure that IGJC2024 continues the success of this highly anticipated and long-running forum by facilitating the dissemination of new discoveries, fostering collaboration, and attracting trainees from diverse backgrounds supporting early-career investigators in the fields of connexin, pannexin, and innexin biology.

NIH Research Projects · FY 2024 · 2024-07

ABSTRACT Aspergillus fumigatus is the main etiological agent of invasive aspergillosis (IA). IA primarily affects immunocompromised patients and carries a mortality rate as high as 60%. Due to the significant increase in the immunocompromised patient population and the emergence of azole-resistant A. fumigatus, a critical understanding of A. fumigatus biology is needed to improve patient outcomes. Caspofungin, which targets fungal cell wall synthesis, is a second-line therapy for invasive aspergillosis. However, this antifungal is fungistatic rather than fungicidal. Comprehending how A. fumigatus responds to caspofungin can lead to a much-needed breakthrough, improving the caspofungin treatment success. Septins are a conserved family of GTP-binding proteins. Septins interact with each other to form higher- order structures and recruit other proteins. Septins play roles in recognizing micron-scale plasma membrane curvature, cytokinesis, cell cycle progression, and response to cell wall stress. The overall aim of our R01 application is to determine the molecular mechanism that contributes to the septin-dependent fungal response to caspofungin. We hypothesize that septin AspB mediates Aspergillus fumigatus fungistatic response to caspofungin. Our hypothesis is supported by 1) an increase in susceptibility to caspofungin after deletion of core septin genes in various fungal pathogens, 2) the increase in the number of visible septin structures after exposure to caspofungin, 3) protein pulldowns that show septin complex together with components of the cell wall integrity pathway, and 4) preliminary proteomics analysis that shows differential septin-protein interactions after exposure to caspofungin. We will test our central hypothesis in three aims: Aim 1. Determine the role of the septin cytoskeleton in response to caspofungin, Aim 2. Define septin-protein interactions after caspofungin exposure and Aim 3. Elucidate the role of the septin cytoskeleton in the cell wall integrity pathway. Completing this grant will close a critical gap in understanding how fungal pathogens respond to caspofungin and how septins facilitate this. With this knowledge, novel therapeutic approaches can be designed to ultimately improve the outcome of caspofungin treatment.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Human mimicry, referred to as synchronization or the "chameleon effect," involves unconsciously imitating gestures, behaviors, facial expressions, speech patterns, and even the emotions of others. This phenomenon is pivotal in social interactions because it fosters social bonding, group cohesion, and cooperation by influencing how others are perceived. Mimicry fosters conscious empathy, aiding individuals in understanding and empathizing with the emotions and intentions of others. Meanwhile, animals also synchronize behaviors, but there are no studies on the underlying circuit mechanisms due to the lack of laboratory paradigms. We have developed a method to quantify the synchronization of emotional responses in mice and found that mice synchronized conditioned freezing, which required the integration of visual social stimulus and the auditory emotional cue. In both sexes, the positive synchronization required the ventral hippocampus (vHPC), its inputs to the amygdala, and vision, suggesting that vHPC processes the partner's visual social stimuli and then feeds it into the auditory fear circuit at the amygdala level to achieve synchrony by modulating freezing. Surprisingly, vHPC inactivation caused sex-specific effects: male dyads lost the positive synchrony, but the females switched from positive to "negative synchronization" and actively avoided aligning their freezing bouts. It suggests fundamental sex differences in the socio-emotional integrator. We will test a hypothesis that a female-specific superior colliculus-mediated alternative visual stream to the auditory fear circuit drives negative fear synchrony, competing with the vHPC stream for positive synchrony. We will use chemogenetic disconnection in Aim 1 to identify the specific synapses underlying negative synchrony and ex vivo recording in Aim 2 to identify sex differences in the synaptic properties of the superior colliculus visual pathway. This study will generate fundamental knowledge about sex differences in the integration of social visual information and auditory emotional information and how the brain organizes even simple behaviors through competing circuits.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Osteosarcoma (OS) is a common and devastating primary bone tumor affecting both humans and canines. Histotripsy is an emerging non-thermal ablation modality that uses high-pressure focused ultrasound pulses to induce acoustic cavitation and liquify target tissue with high precision. Our team has pioneered the development of histotripsy for bone tumors with critical ex vivo and in vivo studies using excised canine OS tumors and pet dogs with spontaneous OS, respectively. Dogs have an increased incidence of OS with high similarity to human OS, allowing the pet dog to serve as a valuable comparative oncology model. This proposal seeks to further the development of histotripsy for OS treatment by 1) investigating the treatment parameters & strategies needed for complete ablation in ex vivo canine OS, and 2) testing these strategies in pilot clinical trials with acute and chronic follow-up of pet dogs with histotripsy-treated OS. Throughout these experiments, I will be trained in vital skills in bio-engineering and clinical oncology, including transmit-receive circuitry (Dr. Tim Hall at University of Michigan), histopathological analysis of ablation (Dr. Sheryl Coutermarsh-Ott at Virginia-Maryland College of Veterinary Medicine), and radiological assessment of ablation (Dr. Tim Ziemlewicz at University of Wisconsin). The results of this work will overcome previous technical & engineering barriers in achieving complete ablation of bone tumors and will provide crucial safety & feasibility data to guide and inform the clinical translation of histotripsy as a non-invasive limb salvage treatment for OS.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Malaria is a major public health burden, causing over a half million deaths and two hundred million clinical episodes annually. During the pathogenic, asexual erythrocytic stage, the malaria parasite scavenges the fatty acids (FA) it needs for lipid synthesis from the host’s plasma. This metabolic requirement is a vulnerability that could be exploited to block parasite replication. Two parasite acyl- Coenzyme A synthetases, ACS10 and 11, likely play key roles by activating fatty acids to acyl-CoA thioesters, although little is known about their specific roles. Mutations in both enzymes have been associated with resistance to anti-malarial compounds, with ACS10 shown to be a direct target, and an ACS inhibitor is currently undergoing phase I clinical trials. Thus, a deeper understanding of the roles of ACS10 and 11 could accelerate drug discovery efforts. We hypothesize that ACS10 and 11 each make important contributions to parasite FA uptake through distinctive specificities. In this proposal, we will develop a new approach, termed “FA alkyne profiling”, for the systematic analysis of FA uptake and lipid biosynthesis in P. falciparum and will employ it to gain insights into the roles and specificities of ACS10 and 11 and the effects of ACS inhibition. In Aim 1, we will optimize FA alkyne profiling in P. falciparum using six structurally-diverse fatty acid alkynes that together represent three- quarters of P. falciparum fatty acids. Each FA alkyne probe will be validated in competition assays with natural FAs and in parasite viability assays. These studies will provide a quantitative basis for exploring the effects of perturbations in parasite fatty acid metabolism. In Aim 2, we will employ FA alkyne probes to interrogate the physiological roles of ACS10 and 11. Parasite lines capable of inducible knockdown of ACS10 or 11 will be profiled to establish the effects of enzyme depletion on FA alkyne utilization. We will also use FA alkyne profiling to investigate the effects of two validated ACS inhibitors with anti-malarial activity. These studies will provide insights into the physiological roles of specific parasite ACSs and will establish a general framework for investigating he effects of inhibition or knockdown of key lipid metabolic enzymes.

NIH Research Projects · FY 2026 · 2024-07

A pulse-driven micropump for transdermal drug delivery PROJECT SUMMARY/ABSTRACT Ambulatory infusion pumps are an increasingly prescribed therapy modality for cancer and diabetes patients, post-operative pain management, and more. They allow patients to receive necessary medication infusions, but are often bulky, tethered to the user with tubing, and associated with pain at the insertion site. These shortcomings make drug regimen adherence harder for infusion pump users, particularly those with chronic conditions. To eliminate barriers to infusion therapy adherence, there is a critical need for compact, lightweight infusion devices that don’t impede physical activity. Our long-term goal is to improve infusion therapy adherence by developing featherweight arterial pulse-driven infusion pump technology that eliminates the need for painful cannula insertions and motors and batteries to facilitate pumping. Our overall objectives in this project, the next steps toward our long-term goal, are to (i) characterize the infusion rate capabilities of a prototype pulse-driven micropump based on our recent advances in bio-inspired microfluidic pump technology, and (ii) tune the micropump design to deliver a set of target infusion rates within its range by way of physical experiments, computational modeling, and AI-guided optimization. Our central hypothesis is that our pulse- driven microfluidic pump technology, which mimics natural biological function, can provide drug delivery rates appropriate for clinical applications like ambulatory chemotherapy for a wide range of users with varying arterial pulse profiles. Our hypothesis is based on preliminary data demonstrating the proof-of-concept that our prototype micropump can be powered by the human radial artery pulse and produce flow rates appropriate for chemotherapy and insulin delivery. The project’s rationale is that, before the proposed pulse-driven micropump can be developed for clinical use, its capabilities must be characterized, allowing its design to be optimized for specific applications. To attain these objectives, the following specific aims will be pursued. First, we will determine the dependence of the micropump flow rate on device design parameters and arterial pulse characteristics using in vitro and in vivo tests with rapid prototype micropumps produced using microfluidic fabrication techniques and 3D printing; next, we will develop a 3D finite element model (FEM) of the prototype micropump; finally, we will use an evolutionary algorithm along with human subject and porcine skin transdermal flow rate testing to develop an initial set of 6 wearable infusion patch pump designs integrated with microneedle arrays. Upon project completion, we expect our contribution to be a featherweight pulse-driven infusion pump capable of producing a wide range of infusion rates that can be integrated into low-cost, disposable infusion devices the size of a nicotine patch, or in state-of-the-art closed-loop and implantable infusions systems, greatly reducing the footprint of both types of devices. This advance in infusion technology is expected to increase users’ adherence to their drug regimens, reduce risk of diabetic hyperglycemic crises, and improve quality of life for many patients. The technology has an additional potential application in self- administered vaccine patches.

NIH Research Projects · FY 2026 · 2024-07

Summary PRMT5 is an essential enzyme and the cell’s primary enzyme capable of symmetric dimethylation of arginine residues. PRMT5 is a therapeutic target in cancer and neurodegenerative diseases. PRMT5 methylation of multiple protein substrates regulates a host of important cellular pathways and processes including transcription, chromatin dynamics, mRNA splicing, and translation. While PRMT5 activity is required for cell and tissue survival, which function(s) or substrate(s) of PRMT5 are required for general cell maintenance or involved in cancer and neurodegenerative diseases has not been elucidated. Further, while PRMT5 substrate proteins are strongly enriched for RNA binding and RNA regulatory pathways, the biochemical effects of arginine dimethylation have not been resolved. Specifically, methyl-Arg increases the size, hydrophobicity, and charge distribution, and decreases the H-bonding potential of substrate arginines but the mechanics of how the Arg-methyl groups affect PRMT5 substrates’ structure/function and subsequent cell survival are not known. In this project, I will explore how PRMT5 methylation of substrate proteins affect their ability to (1) bind to mRNA, (2) bind to other proteins/form higher order protein complexes and (3) affect substrate-dependent processes such as mRNA splicing. We will identify how substrates are recruited for methylation by PRMTs and what the downstream biochemical function is in order to better understand PRMT5 essential functions in normal cells and disease. Achieving a therapeutic window where select PRMT5 activities can be blocked without disrupting normal cell function is critical for targeting this essential enzyme. Thus, understanding the mechanisms of PRMT5 biology has the potential to inform our understanding of an important posttranslational modification on multiple intracellular targets and to guide future efforts in developing modulators of PRMT activity in disease settings. The discovery of novel sites on PRMT5 or on substrates may provide new therapeutic targets that would allow for targeting select PRMT5 function in disease while preserving the normal healthy cells and overcoming issues of toxicity observed with broad, catalytic PRMT5 inhibitors. We have previously defined the first mechanism of substrate recruitment to PRMT5 through the conserved, colinear PRMT5 binding motif using a computational tool to discover enriched protein motifs. We have predicted a new binding site for several additional PRMT5 substrates including ZNF326 – a substrate that is consistently methylated in all cells tested and that regulates mRNA splicing. We will determine how substrates get recruited to PRMT5 (and other PRMT family enzymes) for methylation and their cellular and biochemical outcomes as we establish a comprehensive research program devoted to understanding how enzymes select their substrates and performing early validation of their therapeutic potential. Overall, this project will dissect the cellular consequence of substrate arginine methylation by PRMT5 and from this we can uncover how therapeutic PRMT5 inhibitors will affect disease and normal tissue.

NIH Research Projects · FY 2026 · 2024-06

Project Summary/Abstract The human metabolome remains mostly unknown, presenting a major bottleneck in the discovery of new biological mechanisms because structures are needed to predict function. There is a critical need for high-throughput, experimental strategies to characterize these unknowns. The overall goal of this project is to characterize unknown small molecules in humans using a combination of organic synthesis, mass spectrometry and public data mining. This proposal focuses on the synthesis and detection of two classes of small molecules critical to human physiology – neurotransmitter derivatives and sphingolipids. For the proposed projects, a strategy called reverse metabolomics will be used. Typically, in an untargeted metabolomics experiment, compounds are detected first, prioritized based on biological significance, then structurally identified. In the proposed reverse metabolomics experiments, however, the process is flipped. Compound classes of interest are first identified and synthesized, then their spectra are searched for in public metabolomics data to see if they are found in humans and if so, where. The proposed work will generate large MS/MS libraries of previously unidentified metabolites and all data will be made public. Additionally, a novel catalytic method for the one-step divergent synthesis of sphingolipids will be developed. While the proposed studies focus on two specific types of molecules, this strategy can be readily adapted to study other classes of biological molecules. Ultimately, this research enables the identification of new potential biomarkers, therapeutic targets, and pathogenic mechanisms.

NIH Research Projects · FY 2025 · 2024-06

Project Summary Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and sterility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. Brucella spp. are considered threats as potential biological weapons. Importantly, antibiotic treatment against brucellosis is prone to disease relapse, and there is currently no safe and effective vaccine to protect humans against infection with Brucella. The brucellae are intracellular pathogens that reside within immune cells called macrophages where they replicate in a specialized compartment, and the capacity of Brucella to survive and replicate within macrophages is essential to their ability to cause disease. Over the last few years, our laboratory has characterized genetic pathways that are critical for the intracellular survival and pathogenesis of Brucella strains, and specifically, we have identified small regulatory RNAs (sRNAs) that are essential for Brucella virulence. Preliminary experiments have revealed the presence of more than 20 novel sRNAs in B. abortus, and we have identified one of these sRNAs, called Bsr7 (for Brucella small RNA) that is required for the ability of the bacteria to withstand outer membrane stress. Given the strong connection between Bsr7 and the integrity of the cellular envelope, we hypothesize that deletion of Bsr7 will lead to significant attenuation of B. abortus in both macrophage and animal models of infection. Additionally, we hypothesize that Bsr7 is produced under biologically relevant conditions, such as acidic pH, oxidative stress, nutrient limitation, and/or diminished oxygen. Moreover, it is hypothesized that Bsr7 regulates the expression of genes important for the infectivity of B. abortus. Therefore, we plan to characterize the biological and regulatory functions of Bsr7, and in the end, the information gleaned from these studies may be used to develop new therapeutic and vaccine strategies against human Brucella infection.

NIH Research Projects · FY 2025 · 2024-06

Project Summary Zika virus (ZIKV) causes congenital ZIKV syndrome in infants exposed in utero following maternal ZIKV infection via mosquito bite or sexual transmission during pregnancy. It is estimated that 3-23% of ZIKV cases are due to sexual transmission. More than half of ZIKV-infected men shed ZIKV in semen, and some men shed ZIKV in semen for up to 6 months post-disease onset. In human semen, ZIKV infects several cell types including epithelial cells and immune cells. Using a mouse model, infection of epididymis epithelial cells has been shown to be critical for acute ZIKV shedding in semen. The role of immune cells in ZIKV shedding in semen is unknown, though we detected persistent ZIKV infection within the epididymal lumen, rather than the epididymis epithelium, suggesting immune cells may be critical for persistent ZIKV shedding. The most closely related virus to ZIKV, Spondweni virus (SPOV), is poorly shed in semen despite dissemination to the male reproductive tract. ZIKV containing the prM/E structural genes from SPOV is shed in semen at significantly lower levels during acute infection. The long-term goal of this project is to understand the mechanisms of ZIKV shedding in semen and sexual transmission. The objectives of this study are to identify the viral factors and cell types that contribute to acute and persistent viral shedding in semen. The hypothesis is that ZIKV shedding in semen is driven by viral structural genes and persistent infection of immune cells in the male reproductive tract. Two specific aims will address this hypothesis: 1) Determine the role of viral structural genes in acute ZIKV shedding in semen; and 2) Determine the role of immune cells in persistent ZIKV shedding in semen. In the first aim, we will use our reverse genetics system to generate a reverse genetics system for SPOV and a chimera containing ZIKV prM/E structural genes within SPOV. Viruses will be used to infect male mice, and viral shedding in ejaculates will be measured. Cell types differentially infected within the epididymis epithelium will be identified. In the second aim, macrophages will be depleted from ZIKV-infected mice during acute and persistent infection. Viral shedding in semen and infection of the epididymis will be measured. The research proposed here is innovative because it assesses ejaculates, which are biologically relevant samples for sexual transmission, and a novel role for immune cells in ZIKV sexual transmission. Upon successful completion of the proposed research, the anticipated contribution of this work will be the identification of the flavivirus factors that dictate viral shedding in semen and the cell types that contribute to viral shedding in semen. This contribution is expected to be significant because it will increase our understanding of how viruses are sexually transmitted.

NIH Research Projects · FY 2025 · 2024-05

Project Summary Staphylococcus aureus is a bacterial pathogen that provokes a diverse array of human diseases, ranging from mild skin lesions to invasive and life-threatening infections. The success of S. aureus as a pathogen is attributed to its resistance to antibiotic therapy and production of surface adhesins and glycopolymers, as well as secreted proteins such as cytolysins, superantigens, and proteases, many of which play vital roles in immune evasion. S. aureus also produces extracellular membrane vesicles (MVs) that encapsulate cargo that includes lipoproteins, nucleic acids, glycopolymers, adhesins, and exoproteins, including proteases and pore-forming toxins. Numerous studies have characterized MV production from cultures of various S. aureus isolates and demonstrated the multiple biological activities of MVs in vitro and in vivo. However, the generation of MVs in vivo during staphylococcal infection remains unproven, resulting in questions about the biological relevance of MVs and their contribution to microbial virulence. The long-term goal of this project is to understand the biological roles of MVs in the pathogenesis of staphylococcal infections. The objective of this proposal is to characterize the production of S. aureus MVs in vivo to better understand their contributions to disease. The central hypothesis is that release of MVs is a critical virulence mechanism by which S. aureus targets host cells while protecting the MV cargo from destruction by environmental factors. This expectation is based on published findings showing that S. aureus MVs purified from in vitro cultures encapsulate a variety of virulence determinants with pronounced biological activities. Preliminary data in this application show that S. aureus MVs are generated in a murine air pouch infection model. This project will establish and optimize methodology for the purification and analysis of S. aureus MVs recovered from infected animals. The specific aims of the application are to: (1) Characterize the production of S. aureus MVs during infection – murine air pouch and pneumonia models. The pneumonia model will allow a more comprehensive understanding of S. aureus MV production in a model resembling human disease. The protein composition of S. aureus MVs generated in vitro vs. in vivo will be characterized by LC- MS/MS. If the MV protein content differs in vitro vs. in vivo, RNA-Seq analysis will be performed on S. aureus bacteria cultivated in vitro vs. in vivo to correlate gene expression with MV protein cargo. (2) Evaluate the biological effects of “in vivo” MVs on the host. To uncouple the biological responses to MVs from those elicited by bacterial cells, physiologically relevant numbers of MVs purified from air pouch and bronchoalveolar lavage fluids will be used for (a) in vitro cytotoxicity assays and (b) to inoculate naive mice. The animals will be monitored for leukocyte influx, cytokine levels, and tissue pathology. To accomplish these goals, a multidisciplinary approach will be taken that combines microbiology, molecular biology, biochemistry, immunology, and animal models. Knowledge gained from these studies will deepen the currently limited understanding of the role that MVs play in bacterial pathogenesis, especially those produced by Gram-positive pathogens.

NIH Research Projects · FY 2026 · 2024-05

Summary The goal of this project is to develop a novel histotripsy device for non-invasive treatment of heterogeneous osteosarcoma tumors (OS). OS are malignant bone tumors that develop in both children and adults. Long-term survival rates for patients with metastatic and non-metastatic OS are around 20% and 70%, respectively, highlighting the devastating nature of this disease. Limb salvage surgery or amputation are first-line treatments for primary appendicular OS, but these remain associated with high complication rates and decreased mobility and function. Novel treatments for OS are needed to improve outcomes. Histotripsy is a non-invasive ultrasound therapy that mechanically ablates tumors into acellular debris via controlled acoustic cavitation. Histotripsy can be tissue-selective, where neurovascular bundles, vessels, and bone can be preserved while the tumor is completely disrupted to acellular debris. Histotripsy appears to be well-suited for OS with the ability to serve as a non-surgical limb salvage treatment option, with promising results noted in preliminary studies. Despite this promise, OS treatment with histotripsy presents unique challenges: 1) OS tumors exhibit high inter- and intra- tumoral heterogeneity, with varying proportions of lytic and proliferative bone and soft tissue tumor proliferation, requiring optimized treatment strategies for complete and uniform ablation of all OS phenotypes. 2) OS tumors grow close to critical structures. While histotripsy has been shown to be tissue-selective for other applications, there remains a need to develop methods for selectively ablating OS tumors while preserving healthy bone, nerves, vessels, and connective tissue. 3) Histotripsy is typically guided by real-time ultrasound imaging, which is not feasible for a subset of OS tumors due to bone obstruction, requiring improved targeting and monitoring techniques. In this proposal, we will develop an integrated image-guided histotripsy system for the precise targeting and ablation of OS tumors. The system will consist of a phased array transducer with transmit-receive capability for 3D cavitation monitoring, image-fusion targeting (CT/MRI), and a fully automated robotic treatment technique with strategies for achieving uniform and complete ablation of heterogeneous OS tumors. We propose the following three aims. Aim 1: Design and construct an integrated histotripsy OS system consisting of an array transducer with transmit-receive capabilities for 3D cavitation imaging and image-fusion targeting. Aim 2: Develop patient specific treatment methods with optimized parameters for complete, uniform, and tissue- selective OS ablation. Aim 3: Test the in vivo safety and efficacy of the integrated OS system (Aim 1) and optimized parameters for uniform and safe treatment of OS (Aim 2) in canine OS patients at the Virginia Tech Animal Cancer Care and Research Center. This project will result in a human prototype histotripsy system with unique treatment and monitoring capabilities as well as parameters optimized for OS. The canine patients are the best large animal model to test the safety and efficacy of histotripsy and accelerate clinical translation, and the veterinary treatment is another application for histotripsy OS therapy before the FDA approval of human use.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Lead (Pb) poisoning is a persistent health issue in the United States due to legacy contamination from Pb-based sources such as paint, plumbing, and gasoline. Children are most susceptible to Pb poisoning, which causes persistent cognitive deficits and low IQ. The specific mechanisms by which Pb poisoning impacts development are not fully understood, however. Previous research showed that Pb impacts several thyroid hormone (TH)-related processes, including decreases in expression of TH distributor proteins (THDPs) in the choroid plexus. This is an important issue because TH is a critical regulator of brain development; disrupted TH signaling in utero leads to smaller brain size and mental retardation. The impact of Pb-induced THDP impairment on development has not been fully tested. Given the impact of Pb on THDP expression, our central hypothesis is that Pb poisoning impairs TH distribution into the brain and compromises TH-dependent developmental mechanisms. TH regulates important aspects of brain development in both humans and amphibian, in some surprisingly similar ways. The overall rationale of this proposal is that TH is the key driver of metamorphosis in frogs, which makes tadpoles ideally suited to address how Pb dysregulates TH-dependent mechanisms of development. We will use Xenopus laevis tadpoles to assess the impact of Pb on the expression and function of THDPs in the choroid plexus and its effects on TH-sensitive cellular and molecular mechanisms brain development. The central hypothesis will be tested in three specific aims: 1) To determine the effects of Pb poisoning on expression of THDPs, TH-sensitive genes, neurogenesis, neuronal differentiation, and spine maturation using time-lapse in vivo imaging techniques. 2) To test if knockdown of THDP expression in the choroid plexus mimics the effects of Pb poisoning. 3) To test if overexpression of THDPs in the choroid plexus can counter the effects of Pb-poisoning on TH- dependent mechanisms of brain development. The outcomes of this research will test a significantly understudied mechanism by which Pb is hypothesized to compromise development. These protocols will serve as a new, unique set of sensitive end points to evaluate not just heavy metals but any chemical suspected of disrupting THDP expression and function. This proposal is innovative because it uses advanced techniques that have not been previously used in animals to address the issue of developmental Pb neurotoxicity. This project is significant because it will conclusively test the degree to which Pb affects development via dysregulation of TH-signaling and therefore may lead to alternative/complimentary therapies for counteracting Pb-poisoning in children.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Changes in ion channel biophysics and function due to mutation or disease-associated remodeling are frequently assessed in isolated cells; yet translation of these mechanistic changes for therapeutic target development is difficult and often fails due to the inherent lack of tissue-scale regulation that is missing in isolated cell experiments. Sodium channel gain-of-function is a critical example of this translational challenge, with well-identified ion channel biophysical dysfunction (i.e., gating); yet an estimated 16- 64% of congenital sodium channel gain-of-function patients present without an electrocardiographic phenotype. Our prior work demonstrated a proof-of-concept that modulation of ion concentrations in extracellular nanodomains can conceal or unmask this gain-of-function, and these dynamics are an inherently tissue-scale phenomenon, as the sodium channel enriched nanodomains adjacent to gap junctions at the intercalated disc. The current proposal seeks to demonstrate that this concept has significant potential for therapeutic target development and pre-clinical predictive assessment. Preliminary data demonstrate that extracellular sodium and potassium (through co-regulation by intercalated disc- localized potassium channels) can modulate the presentation of sodium channel gain-of-function in a manner that depends on intercalated disc structure. We propose computational simulations (novel structurally detailed tissue models) and experiments (isolated myocytes, ex vivo hearts, and in vivo genetically-modified mice and peptide treated guinea pigs) to test the hypothesis that sodium and potassium concentrations can serve as critical biomarkers that are co-factors for risk of electrophysiological dysfunction in the setting of sodium channel gain-of-function and concomitant perinexus expansion. While ion concentrations are already established biomarkers in clinical care, our proposal will test the hypothesis that so called ‘normal’ ranges may indeed be pathological in patients with sodium channel gain-of-function. Indeed, the standard collection of these biomarkers is an asset for future work that will seek to identify patients at greater risk for electrophysiological dysfunction. Upon successful completion of these aims, we will produce new mechanistic understanding of the manifestation of sodium channel gain-of-function, which will demonstrate a mechanistic equivalency between detection and therapy.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY AND ABSTRACT Rationale: Despite novel chemotherapies have significantly improved the prognosis of melanoma patients, drug resistance occurs inevitably and tumor progression becomes inexorable, presenting an unmet and urgent need for novel therapeutic intervention. Most melanomas harbor oncogenic BRAFV600E and are responsive to the BRAFV600E-specific inhibitor dabrafenib and the MEK inhibitor trametinib; however, patients eventually acquire resistance mainly due to MAPK reactivation and/or the counteraction by phosphoinositide 3-kinase (PI3K). While substantial efforts have been made to mitigate therapy resistance using PI3K inhibitors; there has been a lack of success as no PI3K inhibitors are currently capable of serving for this purpose. PI3K has four functionally divergent catalytic kinases PI3Kα/β/δ/γ. Drugs that block all four kinases or individual PI3Kα/δ/γ have shown clinical benefit, but elicited notable metabolic or immune-related toxicities. Such clinical challenge in drugging PI3Kα/δ/γ has fueled interest in PI3Kβ therapies; however, PI3Kβ ATP competitive inhibitors GSK2636771, TGX-221, and AZD6482 are not clinically effective, notwithstanding their promising preclinical anti-cancer activities. This is perhaps because ATP competitive inhibitors interact with well-conserved residues at motifs pivotal for executing kinase activity in PI3Kα/β/δ/γ, making them not as PI3Kβ-selective as desired. Rising to this challenge, we have recently identified an 18-residue motif dubbed β18 that occurs only in PI3Kβ, but not in PI3Kα/δ/γ. Selectide-18 bearing β18 and a cell- penetrating peptide distorts PI3Kβ complexes, inactivates PI3Kβ, slows down the growth of BRAFV600E melanomas expressing high levels of PI3Kβ and low levels of the PI3K-antagonizing phosphatase PTEN (designated as PI3Kβhyper), and sensitizes therapy-resistant PI3Kβhyper melanoma cells to dabrafenib/trametinib. Our further in-silico analyses have identified Selectide-9, a short version of Selectide-18 which only contains 9 residues derived from the S18 motif, but still retains the same cytotoxic activities as Selectide-18. Hypothesis: We hypothesize that Selectide- 9 selectively degrades and inactivates PI3Kβ, thereby overcoming therapy resistance in PI3Kβhyper melanoma. Specific Aims: We will use approaches established in the PI’s laboratory to test the above hypothesis. In Aim 1, we will test how Selectide-9 degrades and inactivates PI3Kβ selectively using biochemical and imaging techniques. In Aim 2, we will test whether Selectide-9 is superior to ATP competitive inhibitors in overcoming therapy resistance using two human BRAFV600E patient-derived xenograft lines in immunodeficient mice. The WM4701-6673 line has low levels of PI3Kβ and the recurrent/therapy-resistant WM4701-7329 tumor is derived from WM4701-6673 treated with a BRAFV600E inhibitor. We will determine if WM4701-6673 is sensitive, whilst WM4701-7329 is resistant, to dabrafenib and trametinib and if Selectide-9 is more potent than GSK2636771 in restoring the sensitivity to dabrafenib and trametinib. Moreover, we will identify gene signatures defining therapy-resistant PI3Kβhyper melanomas using single-cell RNA sequencing. Impact: Our work will lay the groundwork to support future studies to gain deeper insights into therapy resistance in melanoma and identify an innovative approach to circumventing this resistance.

NIH Research Projects · FY 2026 · 2024-03

Project Summary: The encoding and consolidation of hippocampus-dependent memories requires temporal coordination of pyramidal neurons in the CA1 region. Specifically, populations of pyramidal neurons organize into sequences representing experience during theta and sharp-wave ripple oscillations. Disruption of the temporal coordination of pyramidal neuron spikes during SWRs, as occurs in pathologies such as epilepsy, results in memory deficits. The cellular and synaptic mechanisms underlying pyramidal neuron spike timing during SWRs with mnemonic functions are largely unknown. Local interneurons, via GABAergic inhibition, are known to regulate CA1 pyramidal neuron spike timing in SWRs. However, there are approximately twenty types of such interneurons, each with distinct anatomy and physiology. How each of these interneurons contributes to spiking activity and memory is unknown. The interneurons hypothesized to have the most precise control over pyramidal neuron spike timing are the axo- axonic (or chandelier) cell, which exclusively synapses directly onto the axon initial segment (AIS), and basket cells which densely synapse on the soma. The functions of these cells in SWR dynamics, replay and spatial memory formation have remained poorly understood, as there have been no reliable markers to genetically access these neurons. In vivo investigations into the effects of axo-axonic and basket cells on pyramidal neuron dynamics in SWRs, replay and their role in spatial memory formation is thus needed. In this proposal, we aim to investigate the role of axo-axonic and basket cells in mnemonic functions of hippocampal circuits, specifically SWRs and replay, and spatial memory formation. Cre drive lines combined with viruses encoding Cre-dependent opsins enable us to manipulate specific interneuron activity in behaving animals. The overarching goal of this proposal is to understand how the unique physiology of axo-axonic and basket cells controls hippocampal circuit activity and pyramidal neuron coordination in network oscillations in support of spatial memory formation.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Nutrient sensing pathways (mTOR, AMPK, sirtuins) are core components underlying the aging process, linking post-translation modifications critical to cellular function to environmental factors. To date, research in this area has largely focused on interventions such as caloric or protein restriction that drive lifespan or slow aging-related morbidities. An emerging area of research derived from these intervention studies highlights the potential of fat catabolism, the lipolytic degradation of triacylglycerol stored within lipid droplets (LDs), as a major factor during the aging process. Despite accumulating evidence for a beneficial role of lipolysis, the mechanism that links lipolysis to healthspan is poorly understood. Dr. Charles Najt recently identified the LD protein perilipin 5 (PLIN5) to be the critical link between lipolysis and the nutrient sensor SIRT1; loss of PLIN5 ablated adipose-triglyceride lipase (ATGL)-mediated activation of SIRT1. Our published and preliminary data show that in response to cAMP/PKA signaling, which is driven by fasting or caloric restriction (CR), PLIN5 binds and transports monounsaturated FAs (MUFAs) produced from lipolysis to the nucleus. Once in the nucleus, MUFAs liberated from PLIN5 allosterically activate SIRT1. These results provide an underlying mechanism explaining the growing body of literature that has linked MUFAs to improved healthspan. Yet, while CR or intermittent fasting, interventions shown to increase healthspan, increase PLIN5 expression, little is known about the direct role of PLIN5 in healthspan or longevity. A few studies indicate Plin5 expression peaks around middle age, slowly decreasing over time or drastically decreases in oxidative tissues during metabolic disease, yet the cause of these changes or the impact of decreased Plin5 expression is unknown. This is a significant gap in knowledge as PLIN5 has been shown to mitigate metabolic disease but its ability to influence health or lifespan has yet to be established. In the current proposal we aim to fill this gap in knowledge, providing significant results directly linking PLIN5 lipid signaling and metabolic flexibility to healthy aging. We hypothesize that PLIN5 signaling in the nucleus is critical for maintaining metabolic health during aging, whereas breakdown of this signaling axis results in age-related morbidities, decreasing healthspan. Moreover, we propose that maintaining PLIN5 signaling throughout life, will increase healthspan and enhance dietary interventions. This hypothesis is significant as no study to date has determined the role of PLIN5 during aging or what role it has in healthspan promoting interventions such as the Mediterranean Diet, which is considered the ideal diet for healthy aging. By focusing on a novel niche in lipid signaling and directly relating PLIN5 to health and lifespan, we are establishing strong fundamental biology that is extremely relevant to healthy aging. The proposed studies are well-aligned with the funding areas listed as of special interest in aging, allowing the applicant to progress to independent investigator status in a new and growing field.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY/ABSTRACT La Crosse virus (LACV) is one of the most significant arboviral pathogens in North America, annually causing clinical pediatric encephalitis, which are occasionally fatal. Severe neuroinvasive disease cases of LACV most often occur in the Appalachian and Midwest regions of the United States (US) in children under the age of 16, and can result in lifelong neurologic complications, including recurrent seizures, partial paralysis, and cognitive and neurobehavioral abnormalities. Long-term sequelae represent a substantial health and economic burden in terms of cost per patient. No licensed countermeasures, such as vaccines or antivirals, are currently available. To date, only two of the three known LACV lineages, Lineages I and II (principally active within the Appalachian and Midwest regions of the US), are associated with human disease. Lineage III, which is now identified in the Northeastern region of the US, has not yet been associated with clinical disease. My research group recently highlighted the existence of regional variants of LACV in the Northeast (Eastwood et al. 2020) and showed that Lineage III LACV persists locally in multiple species of Aedes mosquitoes. This virus lineage presents an undetermined health threat and investigation is warranted to determine whether the lack of Lineage III-derived cases, to date, is due to under-diagnosis of clinical illness (lack of case recognition), differences in virus virulence, low prevalence of infection in mosquito vectors (effectively limiting human exposure to biting activity in regions where the virus circulates), or limited vector competence by these mosquito species. Our long-term goal is to evaluate the public health significance and risk of emergence of lineage III LACV. We question why human cases have not occurred in the face of entomological risk, and aim initially to study the mosquito vector competency of different lineage strains to determine whether the known presence of LACV in the Northeast is a threat to public health, consistent with the more traditional regions. Having established that Lineage III LACV is circulating in mosquitoes in the Northeastern US, and that isolates of LACV strains present different in vitro phenotypic characteristics, such as altered growth rates, plaque morphologies, and murine virulence (Wilson et al. 2021), this pilot study seeks to address the ability of novel LACV strains to transmit in the vector, with the central hypothesis that this distinct lineage is maintained vertically in local populations of mosquitoes with limited horizontal transmission, resulting in the observed reduced incidence of human disease. We will evaluate both horizontal and vertical transmission of LACV in both a native mosquito species (Aedes triseriatus) and an invasive species (Aedes albopictus) as candidate LACV III disease vectors. The proposed project is an important step to understanding whether Lineage III LACV poses a public health risk to a substantially urbanized geographical region. Subsequent studies will be aimed at understanding mechanisms of lineage differences, climatic impacts in vector competency for LACV, and surveillance of humans and vertebrate hosts to explore the incidence, prevalence, and risk of further emergence of LACV in novel regions of the US.

NIH Research Projects · FY 2025 · 2024-01

Project Summary Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and sterility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. Brucella spp. are considered threats as potential biological weapons. Importantly, antibiotic treatment against brucellosis is prone to disease relapse, and there is currently no safe and effective vaccine to protect humans against infection with Brucella. The brucellae are intracellular pathogens that reside within immune cells called macrophages where they replicate in a specialized compartment, and the capacity of Brucella to survive and replicate within macrophages is essential to their ability to cause disease. Over the last few years, our laboratory has characterized genetic pathways that are critical for the intracellular survival and pathogenesis of Brucella strains, and specifically, we have identified small regulatory RNAs (sRNAs) that are essential for Brucella virulence. Preliminary experiments have determined that one sRNA, called MavR, for MurF- and virulence-regulating sRNA is required for full virulence of B. abortus in a mouse model of chronic Brucella infection. Preliminary work has demonstrated that MavR is a negative regulator of MurF, which is an essential enzyme involved in peptidoglycan biosynthesis. Taken together, these data led us to develop a model for the MavR-MurF genetic pathway that is critical for Brucella virulence, and the work outlined in this application will test several independent hypotheses associated with this important genetic circuit. In the end, the information gleaned from these studies may be used to develop new therapeutic and vaccine strategies against human Brucella infection.

NIH Research Projects · FY 2025 · 2024-01

Project Summary Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and sterility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. Brucella spp. are considered threats as potential biological weapons. Importantly, antibiotic treatment against brucellosis is prone to disease relapse, and there is currently no safe and effective vaccine to protect humans against infection with Brucella. The brucellae are intracellular pathogens that reside within immune cells called macrophages where they replicate in a specialized compartment, and the capacity of Brucella to survive and replicate within macrophages is essential to their ability to cause disease. Interestingly, quorum sensing is an important component of Brucella virulence, but traditionally, quorum sensing is an activity performed by large populations of bacteria, while the brucellae exists primarily in intracellular vacuoles in small numbers. Thus, the Brucella quorum sensing system is atypical to the paradigm of Gram-negative quorum sensing systems, and this application seeks to define novels elements of this pathway. Preliminary work in our group led to the development of a B. abortus strain that is unable to sense the quorum sensing molecule, acyl homoserine lactone (AHL). Deletion of the two genes encoding transcriptional regulators of the LuxR family yielded a quorum sensing “deaf” strain, and this strain will be used to define the Brucella quorum sensing transcriptome in order to identify genetic elements critical to virulence. Additionally, we will test the hypothesis that the quorum sensing “deaf” strain will be highly attenuated in both cellular and animal models of Brucella infection. Finally, it is known that Brucella strains produce a 12 carbon AHL, but no genes are present that encode known AHL synthases. As such, we have developed an unbiased screening strategy to identify the Brucella AHL synthase. In the end, the information gleaned from these studies may be used to develop new therapeutic and vaccine strategies against human Brucella infection.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Lead (Pb) is an everyday contaminant with low level exposure rates of one in every three children worldwide. A major source of exposure to this heavy metal is via drinking water delivered through Pb lines. Children are particularly vulnerable, with an exposure rate of up to 800 million globally, as they absorb Pb differently than adults. Even children exposed to very low levels of Pb often go on to experience intellectual deficits with associative macroscopic changes in the brain. While many of these poor neurological outcomes are ascribed to the ability of Pb to cross the blood brain barrier (BBB), how the affects of Pb on other compartments of the body contribute to an underdeveloped brain remains largely unknown. The gut brain-axis has recently been brought into clinical and preclinical spotlights as accumulating evidence confer bidirectional communication between the two organs. As a collective, the microorganisms/microbiota colonizing the vertebrate gut play a commensal role in training the immune system and promoting healthy brain development. This is particularly evident in cases of gut dysbiosis associated with an array of neurological diseases/disorders of developmental origins. Upon Pb contaminated water consumption, the gut microbiota is amongst the first exposed and are readily altered in species composition which has been reported in a limited number of preclinical animal studies. However, how Pb-induced gut dysbiosis impacts the enteric and central nervous system independent of Pb entry systemically remains elusive. Building on a novel germ-free piglet paradigm developed by our group, we will test the central hypothesis that low level Pb exposure impairs brain development by altering the gut microbiome which in turn negatively impacts distinct central and enteric nervous system cell types. We will employ a multipronged approach to test the following three independent yet complementary Aims. In Aim 1, we will determine the effects of a Pb-altered microbiome on key neurodevelopmental trajectories by recolonizing germ-free piglets with donor gut microbiota exposed to low doses of Pb orally. This will enable us to rule out any direct effects of Pb on the brain via entry through the blood brain barrier. In Aim 2, we will identify and characterize alterations that occur in the gut microbiome after Pb exposure. Using multiomics and several other in vivo and in vitro techniques, we will evaluate changes in microbial diversity, metabolites, and host physiology. Aim 3 will include assessments of both compartments to interrogate direct and indirect Pb-induced alterations in distinct central and enteric nervous system cell types. Completion of these studies will provide mechanistic insights on the cellular, microbial, and systems level and illuminate new aspects of Pb exposure that may lead to novel strategies for targeted therapeutic interventions to improve neurological outcomes in children exposed to Pb.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY In the visual system, retinal axons convey visual information from the outside world to numerous and distinct brain regions. In rodents, one major area that is densely innervated by retinal input is the visual thalamus. Mouse visual thalamus serves as a powerful model system in understanding sensory circuit development, based on its orderly structure and ease of accessibility for experimental manipulation. Visual thalamus, or lateral geniculate nucleus (LGN), is divided into three distinct regions: dorsal geniculate nucleus (dLGN), ventral lateral geniculate nucleus (vLGN), and the intergeniculate leaflet (IGL). Cytoarchitecture and circuitry of dLGN are well-studied, and it is known to be important for classical image-forming vision. vLGN is associated with non-image-forming vision and its complete neurochemistry, cytoarchitecture, and retinothalamic connectivity remain unresolved, raising fundamental questions about its functional role within the visual system. Identifying the structure and function of neural circuits related to non-image-forming vision is crucial for understanding how light exerts its influence on programming an individual’s circadian cycle, mood disorders, fear perception, and eye movement and head movement in response to certain changes in the visual environment. Using state-of-the-art single-cell sequencing and proteomics, we can identify a comprehensive list of the cells in vLGN. Using in situ hybridization, immunohistochemistry, and genetic reporter lines, we found that the subtype-specific laminar distribution of retinorecipient cells in vLGNe is determined during embryonic development. In vLGNe, the retinorecipient portion of vLGN, studies have demonstrated at least six transcriptionally distinct subtypes of inhibitory neurons that are distributed into distinct adjacent sublaminae. Using trans-synaptic viral tracing, we can identify the inputs and outputs of these distinct vLGN cell types with both cell type- and region-specific resolution. By genetically removing visual input, we found that molecular cues and activity from retinal ganglion cells play important roles in the development of cells and circuits in vLGN. Using in situ hybridization, immunohistochemistry, and genetic reporter lines, we can test the role of retinal axons and activity, through retinal and non-retinal morphogens, in vLGN development. Taken together, the proposed studies will not only identify novel subtypes of vLGN cells, but also point to new means of organizing visual information into parallel pathways by anatomically creating distinct sensory channels. This subtype-specific organization may be key to understanding how the vLGN receives, processes, and transmits light-derived signals in the subcortical visual system. Elucidating these pathways will give potentially generalizable principles in how sensory information is organized in the brain, and this would be the first such characterization of non-image-forming visual circuits.

NIH Research Projects · FY 2026 · 2023-11

Summary: Hepatitis E virus (HEV) is an important but extremely understudied human pathogen, causing acute and chronic hepatitis E, high mortality during pregnancy, neurological sequelae, and foodborne hepatitis. According to WHO, estimated 20 million HEV infections occur each year, leading to >3.3 million cases and >44,000 deaths annually. HEV infection is associated with numerous extrahepatic manifestations including a range of neurological sequelae, which is associated with HEV-3 (mainly) and HEV-4 (lesser extent) infection and occur in ~5.5% of HEV-infected patients. HEV-associated neurological sequelae is an emerging clinical problem, but the underlying mechanism(s) of HEV-associated extrahepatic pathogenesis is unknown. In preliminary studies, we showed that HEV infects cells in the neurovascular unit (NVU) including brain microvascular endothelial cells, microglia, and astrocyte, and that HEV causes pathological lesions in CNS in infected pigs with significantly higher levels of proinflammatory cytokines IL-18 and TNF-ɑ in pigs with detectable HEV RNA in brains than in pigs with no detectable HEV RNA in brains. Our pilot study showed that gerbils are highly susceptible to infection by HEV-1, HEV-3 and HEV-4. Our long-term goal is to delineate the mechanisms of HEV-associated extrahepatic pathogenesis. In aim 1, we hypothesize that HEV induces extrahepatic neurological injuries via induction of pyroptosis by infecting NVU cells to release proinflammatory cytokines leading to neuroinflammation and neurological injuries. We aim to: (1) determine the effect of HEV-3-induced pyroptosis in NVU cells; (2) identify the mechanisms of HEV-induced pyroptosis in NVU cells particularly via caspase 1-mediated canonical pathway; (3) identify HEV-specific pyroptosis-associated proinflammatory cytokines in NVU cells; (4) determine whether HEV-induced pyroptosis in NVU cells is HEV genotype-specific. We anticipate that HEV induces pyroptosis in microglia, astrocyte, and neuronal cells; that HEV induces pyroptosis through caspase 1-mediated canonical pathway to produce proinflammatory cytokines to cause acute cell death; that only HEV-3/HEV-4, but not HEV-1, induce pyroptosis in NVU cells. In aim 2, we hypothesize that HEV induces neurological sequelae through injuries of neural cells via induction of pyroptosis in brain and spinal cord tissues. We aim to: (1) infect gerbils with HEV-1, HEV-3 and HEV-4, respectively, to delineate the mechanism of neuroinflammation and neurological injuries attributable to each HEV genotype; (2) define HEV-specific neurological lesions in CNS and correlate neurological lesions with hepatic lesions; (3) identify the mechanism(s) of HEV-induced pyroptosis via the caspase-1–mediated pyroptosis in brain by using a caspase 1 inhibitor (VX-765) in HEV-infected gerbils. We expect that we will identify HEV-specific neuroinflammation and neurological lesions and types of neural cells involved in pyroptosis, that HEV-induced neurological lesions in brain occur mainly in HEV-3/HEV-4, but not in HEV-1, infected gerbils, that HEV induces pyroptosis in brains via the caspase 1-mediated canonical pathway which will involve in activation of inflammasome to release proinflammatory cytokines IL-1β, IL-18, and TNF-ɑ, and that treatment of HEV-infected gerbils with caspase 1 inhibitor VX-765 will reduce the amounts of neuroinflammation and lesions in CNS tissues compared to controls. The information will inform potential therapeutic targets of HEV-related neurological sequelae.

NIH Research Projects · FY 2025 · 2023-09

The emergence and re-emergence of pathogens and their impact on society has reinforced the need for integration and synergy across scientific fields and biological scales in order to advance understanding, predicting, and responding to pathogen spread. Multi-scale mathematical models that consider the timing and length of individual infections when modeling transmission into the population can aid recommendations for optimal interventions. One shortcoming when evaluating data using multi-scale models comes from data scarcity in the expansion stages of the infection and transmission, the differences in data magnitude and frequency at each scale, together with the complexity of the models considered. To determine the source of combined biases in parameter estimation, we will use a combined empirical-theoretical approach for investigating structural and practical parameter identifiability of multi-scale models of infectious diseases that may inform optimal experimental design. The proposed research will facilitate a better understanding of the sources of uncertainty when fitting multi-scale models to multi-scale infectious disease data, with a focus on Usutu and SARS-CoV-2 viruses. By combining empirical and theoretical approaches we aim to determine structural and practical parameter identifiability of multi-scale models, to inform optimal experimental design, and to improve our ability to make predictions and suggest interventions. Our proposal will focus on three major mathematical challenges: (1) Developing methods for improving practical identifiability in within-host systems; (2) Use experimental data to inform development of transmission models; (3) Build a quantitative framework to predict parameter identifiability in multi-scale systems. The overarching goal of the proposed work is to integrate multi-scale mathematical model development and statistical models for data fitting with collection of longitudinal virus titers and probability of transmission data in order to decrease uncertainty and improve results reproducibility. This will ultimately improve our understanding of infection disease transmission and persistence.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract The objectives of my research program are to understand and engineer protein degradation by the ubiquitin-proteasome system—a critical signaling mechanisms that all animals, plants, and fungi use to perceive and adapt to their environment. The ubiquitin-proteasome system acts like the recycling system of the cell, where specific proteins are marked for recycling by ubiquitin and are cut into peptides by the proteasome to be further broken down and made into new proteins. The ability of the ubiquitin-proteasome pathway to remodel a cell’s proteome in a rapid and specific way has perhaps led to its ubiquity throughout the evolution of eukaryotes. The strong conservation of ubiquitin in eukaryotes also makes it a prime candidate for engineering control systems in biology. The ubiquitin- proteasome machinery in humans is frequently implicated in cancers, neurodegenerative diseases, and metabolic disorders among other diseases, due to it is involvement in cell cycle regulation, vascular development, and inflammation, among other critical processes. By improving our understanding of how the ubiquitin-proteasome pathway functions or fails to function, we may uncover new ways to treat or prevent human disease. The ubiquitin-proteasome pathway plays perhaps an even more central role in plants where it is involved in nearly all known plant hormone signaling pathways to coordinate their growth and respond to changes in their environment, including stresses such as pests and pathogens. These chemical hormones activate ubiquitin ligases which trigger degradation of repressive transcription factors leading to activation of hormone-responsive gene transcription. Interestingly, animals and microbes also perceive plant hormones which have wide ranging effects on their physiology. Animals and microbes also produce plant hormones or similar molecules to aberrantly activate ubiquitin-proteasome signaling and manipulate plants for their benefit. Our research aims to understand the molecular mechanism of how information is transferred through these ubiquitin-proteasome signaling pathways and how these hormone-signaling and biosynthesis pathways have coevolved in plants and other eukaryotes. To do this we will use a deep mutational scanning approach enabled by a massively parallel functional assays using a biosensor we recently developed. In parallel, we aim to re-engineer these chemically activated ubiquitin ligases to detect related and potentially novel chemical compounds and to act as rapid metabolic controllers. These molecular tools will improve our ability to control and engineer biological systems and sustainably biomanufacture these plant hormones and related chemicals, including important agricultural and industrial chemicals, nutritional supplements, and pharmaceuticals.