Saint Louis University

NIH Research Projects · FY 2024 · 2024-09

Project Summary Progranulin is a lysosomal and secreted protein that contains multiple cysteine-rich granulin domains. Although its precise molecular function remains unknown, heterozygous progranulin (GRN) mutations are causal for frontotemporal dementia (FTD). FTD is a devastating disease with a mean survival of 7 years from diagnosis and no approved disease-modifying treatment. Since this is a condition of haploinsufficiency, strategies aimed at increasing progranulin levels are feasible therapeutic approaches. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological disorders. ASOs are short, single-stranded sequences of RNA that are designed to bind to their target RNAs and enable highly specific modulation of target levels. We have developed a novel strategy for increasing progranulin levels by using ASOs to sterically block microRNA binding sites in the human GRN mRNA. We have identified ASOs that effectively increase progranulin protein levels in the brains of humanized GRN mice. Building on this, we will: 1) test if ASO treatment improves FTD-associated behavioral deficits and neuroinflammation in humanized GRN mice with haploinsufficiency, 2) define the timing window for effective ASO treatment, and 3) assess potential off-target effects for lead ASOs. Completion of these studies will generate important preclinical data that provide insights into the utility of this ASO-based therapeutic approach for progranulin-deficient FTD.

NIH Research Projects · FY 2025 · 2024-09

Title: Translating research to practice in corrections staff mental health through dissemination of evidence: from pilots to practical programs PROJECT SUMMARY Our collaborative series of research-to-practice (r2p) conference meetings will advance evidence-based interventions to reduce stress and trauma that contribute to chronic health issues among this high-risk workforce. We will utilize r2p methods to advance correctional worker mental health and well-being, through awareness and directed utilization. We will oversee the dissemination of evidence and its conversion to practice in three conference meetings. We intend to listen to worker voices, gain feedback from attending correctional workers and their lived experiences, promote the evaluation of interventions, and inform the usefulness and utilization of evidence-based resources. This innovative annual series builds upon each event and existing best practices, informing the next one to advance interventions. Our efforts to advance r2p in corrections worker health is supported by 15 years of research and locally based practice, and experience building a participatory network of key stakeholders and change agents. Vehicles include hosting three National Corrections Collaborative (NCC) Symposia (CPH-NEW, 2022; El Ghaziri, et al., 2020); and a National Institute of Corrections (NIC) cooperative agreement project to identify resource provision and resource gaps to address organizational stress and trauma among corrections workers. We will optimize conference attendance by echoing our past tandem format that coordinates these NIOSH supported conferences with regular national corrections meetings. Our research has confirmed that serious health issues affect this public safety sector workforce, and that these are exacerbated by violence, trauma, and stress that occur intrinsically in correctional work environments (Jaegers et al., 2022; 2019; Obidoa et al., 2011). Corrections officers are disproportionately burdened by fatalities from violence, by high rates of non-fatal injuries from assaults and involvement in violent events, and by high rates of homicide and suicide in inmates, and (Konda, et. al., 2012). Rates of mental disorders in COs exceed other criminal justice professions and the general public, with 53% of jail officers meeting the criteria for PTSD (Jaegers et al. 2019) and 31% for depression (Jaegers et al., 2020; NIJ, 2016). Our recent NIC investigation revealed the scarcity of intervention research to address the mental health needs of corrections workers. Our scoping literature review for NIC identified only 25 studies in 7 categories that were suitable for building an inventory (policies, practices, peer support, and training) suitable for addressing worker trauma and organizational stress. Through our partnership with NIC we have engaged and expanded the NCC Project Taskforce (NCC-PT) to 41 active members representing correctional workplaces, researchers, unions, and professionals critical to informing conference meeting design and evaluation.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT The CNBD family of channels, encompassing hyperpolarization-activated cyclic nucleotide-gated (HCN) and depolarization-activated KCNH members, plays a pivotal role in shaping the electrical properties of the heart. The modulation of HCN channels presents a promising therapeutic avenue for the management of atrial and ventricular arrhythmias by influencing the activity of the sinoatrial node pacemaker. In addition, the human KCNH2 potassium channel (hERG) gene is linked to long QT syndrome (LQTS), an inherited cardiac condition that substantially escalates the risk of ventricular arrhythmias and sudden cardiac death. HCN and KCNH channels share structural features, most notably their voltage-sensing domains (VSD). However, they display divergent channel gating behaviors. Up to this point, a comprehensive mechanism that adequately explains the diversity in their voltage-dependent gating has remained elusive. In particular, the VSD of HCN channels undergoes distinct conformational changes during activation, whose dynamics, nature of intermediate conformational states, and couplings to other domains remain largely uncharacterized. Moreover, like mutations affecting the VSD of HCN channels, we recently uncovered that several LQTS-linked mutations the S4 helix voltage sensor of hERG channels lead to distinctively altered voltage-dependent activation profiles. The underlying mechanism of these disease-causing gating alterations remains unexplored, yet it is central to our understanding of the mechanism of cardiac diseases related to CNBD channels. The overarching objective of this application is to determine the VSD dynamics of recombinant CNBD channels expressed in human cell lines, using an array of concerted biophysical techniques. We will improve the patch-clamp fluorometry (PCF) method to study the structure-function correlation of cardiac voltage-gated channels, paving the way to new treatments of myocardial diseases. Our primary goals also include studying how pivotal regulatory domains modulate channel gating by altering VSD conformations. To quantitatively assess voltage-dependent structural rearrangements, we will leverage transition metal ion Förster resonance energy transfer (tmFRET), noncanonical amino acid integration, and click chemistry for site-specific fluorescence labeling. This strategy will enable us to precisely translate FRET efficiencies into interatomic distances. In parallel, phasor-plot fluorescence lifetime imaging (FLIM) in conjunction with molecular dynamics (MD) simulations will be used to quantify the underlying structural dynamics. Collectively, the proposed studies will delineate the energetics, allostery, and structural foundations of cardiac pacemaking ion channels.

NIH Research Projects · FY 2024 · 2024-09

The rapid advancement of artificial intelligence (AI) and machine learning (ML) has led to major breakthroughs in molecular structure modeling, particularly for protein structure prediction. However, accurate prediction of RNA tertiary structures remains challenging due to the limited availability of experimentally determined RNA 3D structures and the lack of standardized, AI/ML- ready datasets for training advanced algorithms. Results from the Critical Assessment of Protein Structure Prediction (CASP15) competition indicate that motif-based approaches outperform deep-learning-driven methods for RNA 3D structure modeling. Nevertheless, traditional motif- based methods are limited when applied to RNA molecules for which suitable templates are scarce in existing template libraries. To overcome this limitation, there is a need for ML-driven RNA structure prediction methods that can effectively capture relationships between nucleotides and structural motifs using large-scale RNA sequence and structure data. The integration of RNA motif-based features with AI/ML algorithms shows promise in enhancing RNA structural analysis and prediction accuracy. This proposal will develop an automated RNA motif structure parsing pipeline to generate standardized motif-based feature datasets to support AI- and ML-driven RNA structural analysis. The datasets will facilitate the training and evaluation of advanced ML algorithms and enable a broad range of RNA structure analysis applications. Specific objectives are: 1) develop an automated motif-based feature generation framework for improved RNA structure prediction with machine learning; 2) develop open-source computational workflows for RNA structure analysis using the AI/ML-ready features; and 3) improve sequence-structure modeling in full-length RNA folding by integrating RNA motif features with open-source AI/ML algorithms. The proposed AI/ML-ready features will support computational workflows including RNA motif clustering, identification of 3D motif-motif interactions, and integration with cryo-EM modeling for RNA 3D structure prediction. This project will release publicly available datasets and reproducible ML pipelines to advance fundamental RNA structure research and computational method development. This research aligns with the mission of the NIH NIGMS and the objectives of the AREA program by developing open datasets and reproducible computational workflows for RNA structure prediction.

NIH Research Projects · FY 2024 · 2024-09

Neuropathic pain arises from nervous system injuries due to trauma, disease, or neurotoxin exposure afflicts 15-20 million people in the U.S is very difficult to treat.1-6 Currently available therapeutics include anticonvulsants, antidepressants and opioids; these have limited efficacy and possess many side effects including high abuse liability.4 Novel non opioid based targets are needed for therapeutic intervention. We believe that we have found such target; the Gi/o protein-coupled receptor (GPCR) Gpr34. Our unbiased transcriptomic approach in a rat model of traumatic nerve injury-induced neuropathic pain7 revealed that GPR34 increases in the dorsal horn of the spinal cord ipsilateral to nerve injury. GPR34 is expressed in both humans and rodents and is highly expressed in microglia.8-10 Its primary endogenous ligand is lysophosphatidylserine (LysoPS).11 Little is known about the roles of LysoPS/GPR34 in pain. One study reported GPR34 deletion in the spinal cord reversed nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia.12 Chemical probes for GPR34, especially antagonists, are limited with only on reported antagonists (Takeda).13 We synthesized this compound (SLU-PP-2368) and found it antagonized LysoPS-induced β-arrestin recruitment and reversed mechano- and cold allodynia in two rodent models7,14 with no observable side effects or engaging the endogenous opioid system (preliminary data). This compound has poor physicochemical properties limiting its clinical utility. Using the cryoEM structure of GPR34 bound with a synthetic agonist,15 we synthesized three analogs (SLU-PP-2438, -2439 and -2440) with GPR34 antagonistic activity and improved solubility and stability. Systemic and intrathecal administration of these antagonists reversed behavioral hypersensitivities in two models of neuropathic pain7,14 (preliminary data). LysoPS (i.th.) in rodents recapitulated behavioral phenotypes seen in the nerve-injury models and evoked dose- and time-dependent pertussis toxin-sensitive (Gαi/o-linked) behavioral hypersensitivities that were blocked by GPR34 antagonists. Collectively, these data identify the spinal cord as a potential site of GPR34 antagonist action that contributes to neuropathic pain. The mechanisms engaged by LysoPS/GPR34 signaling that contribute to neuropathic pain are unknown, but studies implicate mitogen-activated protein kinase (MAPK) signaling pathways.11,16,17 Our driving hypothesis is that GPR34 in the spinal cord is a non-opioid based target for therapeutic intervention with GPR34 antagonists and the analgesic actions of GPR34 antagonists result from attenuating MAPK in microglia. We also hypothesize that identifying CNS-active GPR34 hits will allow future development of novel GPR34 antagonists with intellectual property potential as drug candidates for neuropathic pain. Results from our studies are anticipated to develop a strong biological rationale and establish a multidisciplinary team and infrastructure to develop CNS-penetrant selective GPR34 antagonists that will be used for a therapeutics development plan to enable a future application for RFA- NS-21-015, thereby directly addressing RFA-NS-21-029.

NSF Awards · FY 2024 · 2024-09

In the move to digital learning platforms, education has largely left behind the ability for learners to touch and manipulate objects, despite the long tradition of manipulatives being used by educators like Friedrich Froebel and Maria Montessori, and more modern theories of embodied cognition suggesting the range of roles that touch and movement can play in supporting the acquisition of abstract STEM concepts. This shift to digital platforms may especially disadvantage learners with blindness and low vision (BLV), who often rely on audio alone to comprehend ever-more-complicated digital educational content like interactive simulations. This project will use co-design processes to iteratively create haptic input–output devices, TeleTangibles, that can be used to embody STEM concepts in support of the learning of both sighted students and those with BLV. The devices will provide two-way communication with educational PhET simulations, offering a tangible link between the simulation environment and the physical world. The devices can change shape based on input from educational simulations and from remote learners. The devices can also be reshaped manually, providing communication back to the simulation for remote pairs of learners to co-manipulate a shared tangible artifact. They are intended to be used flexibly – on their own, or integrated with a suite of STEM simulations customized to integrate with the devices. Such devices have the potential of expanding STEM participation and access to more learners, both learners with BLV and learners for whom tactile and kinesthetic engagement may offer a novel “way in” to abstract STEM content that may otherwise be forbidding or challenging. Researchers will partner with teachers, BLV students, and sighted students in the 5th to 8th grade to codesign mechatronic technology to explore three research questions: (1) What sensorimotor interactions with mechatronic technologies hold promise for enriching STEM learning for both sighted and BLV learners?; (2) What impact does integrating mechatronic technologies into educational simulations have on sensorially diverse learners’ STEM understandings?; and (3) What impact does the use of networked mechatronic devices have on remote and on co-located collaboration between learners who may or may not have similar sensory profiles? These questions will be addressed via five iterative rounds of Design-Based Research, using mixed methods approaches including semi-structured interviews, interaction analysis, log data analytics, grounded theory video analysis, pre-post STEM content learning assessments, ethnographic observation, micro-genetic analysis of learner conceptual change, and peer conversation transcripts. The technological innovation of the work lies in creating a low-cost, reconfigurable, tangible, haptic interface that can be deployed within a range of learning activities, adding tactile and kinesthetic means of engaging with STEM content; and providing best practices for the design of flexible, multi-purpose haptic systems. The innovation in learning research lies in how the project will evaluate and extend existing theories of embodied cognition by employing them with sensorially diverse learners. This will shed light on an under-studied population and also holds potential for re-evaluating and refining constructs that were developed with sighted populations. This project is funded by the Research on Innovative Technologies for Enhanced Learning (RITEL) program that supports early-stage exploratory research in emerging technologies for teaching and learning. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-09

An award is made to Saint Louis University (SLU) to acquire XtaLAB Synergy-R single crystal diffractometer to enable users to collect high quality diffraction data from single crystals of organic compounds and biological macromolecules with high efficiency and a significantly reduced consumption of energy and water. Thirteen groups from different SLU colleges and one from the Southern Illinois University at Edwardsville rely on the structural facility at SLU for their research leading to breakthrough discoveries impacting society, economy, and human health. Many projects are collaborations with regional and national universities as well as with industrial partners. The advanced low maintenance design and user-friendly interface makes this system particularly suitable for educational purposes to train undergraduate and graduate students, postdoctoral fellows, and high school students and teachers. It will increase the productivity and quality of educational efforts at SLU and other institutions in a greater St. Louis region, will contribute to the teaching of new generation of STEM talents, and will stimulate building partnerships between academia and industry. The new diffractometer will advance cutting edge research of multiple groups from SLU and regional colleges aiming at groundbreaking discoveries in several fields critical for economy and human health. Those include a wide spectrum of research fields such as DNA recombination and repair; blood coagulation; autoimmune complementation; electron transfer; hepatitis B virus replication; discovery of orphan nuclear receptors; retroviral integration; mechanism of prokaryotic heme enzymes; protein translation regulation; novel function of ubiquitin-like membrane proteins; mechanism of plant DNA demythyltransferases; earth-abundant, non-toxic first row transition metal catalysts; glycosylation reaction and the synthesis of complex glycans. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-09

Project Summary/Abstract Calcium ions play critical roles in regulating many biological processes. Mis-regulation of Ca2+ signaling can lead to diseases, and the Ca2+ channels and exchangers are among the most important drug targets. Proper Ca2+ signaling is also vital for the virulence of many pathogenic organisms. Thus, a thorough understanding of the Ca2+ signaling is very important for enhancing human health. The objective of this project is to study Ycx1, a newly discovered protein required for proper Ca2+ signaling in yeast. Disrupting Ycx1 results in a decrease in the levels of cytosolic Ca2+ and the activation of calcineurin, a reduced chitin content in the cell wall, an increased sensitivity to thermal stress, and an impaired respiratory growth. Ycx1 is found in the endoplasmic curriculum (ER) and Golgi but how it works is not known. Phylogenetics analysis suggests Ycx1 is a cation/Ca2+ exchanger (CCX), the newest and the least understood family of Ca2+ exchangers. Our hypothesis is that Ycx1 controls Ca2+ release from the ER and Golgi and regulates cellular metabolism by modulating Ca2+ flow from the ER to mitochondria. To achieve our objectives, three specific aims are proposed: Aim 1: Characterizing Ycx1 and its role in proper Ca2+ signaling. To characterize Ycx1, we will first determine its biochemical activity by expressing the protein in E. coli and examine its ability to transport Ca2+. To test a role of Ycx1 in regulating Ca2+ release from the ER and Golgi, we will use genetically encoded and organelle specific Ca2+ indicators. To determine how Ycx1 itself is regulated, we will characterize known Ycx1 phosphorylation sites, identify proteins that interact with Ycx1, and examine their role in regulating Ycx1 and Ca2+ signaling. Aim 2: Determining the mechanisms by which Ycx1 regulates cellular metabolism. Ca2+ is required for the activity of pyruvate dehydrogenase, and mitochondria and the ER are tethered in close proximity. We hypothesize Ycx1 facilitates Ca2+ flow from the ER to mitochondria to aid in the enzyme activation. To test this, we will determine if Ycx1 is required for mitochondrial Ca2+ and pyruvate dehydrogenase activation during respiratory growth. We will also determine if the tethering of mitochondria and the ER is crucial for the action of Ycx1 in regulating respiratory growth and Ca2+. Aim 3: Characterizing the Ycx1 homolog in Cryptococcus. C. neoformans is a significant pathogen responsible for over 180,000 annual deaths. Through bioinformatics analysis, we identified a Ycx1 homolog in Cryptococcus. This protein may have similar functions to Ycx1 and could represent a novel factor crucial for the virulence of Cryptococcus. To assess this, we will examine the effects of disrupting this homolog in Cryptococcus on calcineurin signaling, chitin level, and sensitivity to 37°C. We will also examine the subcellular localization of this protein and its transporter activity.

NIH Research Projects · FY 2024 · 2024-09

Project Summary/Abstract We request funds to purchase a Bruker Biospin Corp. Q-Band Elexsys E580 Pulse EPR Spectrometer (Pulse EPR) to enable structural dynamics studies of proteins and their complexes and the training of students and researchers. To maximize sensitivity, this instrument is equipped with a 300 W TWT Q-band pulse amplifier, ER5106QT-II resonator, SpinJet AWG, and Stinger cryogen-free low-temperature system. The high sensitivity of the requested pulse EPR enables us to support projects from user communities that are not feasible otherwise. In addition, combined with our current pulse EPR instrument, having access to a second pulse EPR with enhanced sensitivity and capabilities is essential, not only to manage our throughput, but also to serve as a regional pulse EPR facility for the state of Missouri and nearby states. DEER spectroscopy as a powerful pulse EPR technique provides an effective nanometer distance ruler to measure conformational changes in biomacromolecules under relevant physiological conditions. With increasing technological advances in this ensemble-oriented method, atomic resolution structural information can be directly linked to conformational sampling in solution. Thus, mechanistic models for protein function can be obtained and DEER spectroscopy is imperative to establish such models. The instrument will be housed in the Department of Biochemistry and Molecular Biology at the Saint Louis University School of Medicine. The instrument will support basic research of NIH-funded investigators at both Saint Louis University and Washington University in St. Louis. We describe projects from 8 major and minor users with NIH grants that will benefit from the requested instrumentation. The user projects span a range of membrane transporters, unconventional protein secretion, blood clotting processes, biomolecular interactions in DNA repair and recombination, immunothrombosis, regulation of protein translation, antimicrobial peptide transport, ligand-gated ion channels, and membrane protein oligomerization. These projects focus on protein structure, functional dynamics, and interactions, with majority involving low spin concentration samples. Thus, the high- sensitivity Pulse EPR will be critical for these studies. Training for the Pulse EPR will be primarily handled by members of the Dastvan research group who have more than a decade of experience using the instrumentation, sample preparation, and data analysis. Users (graduate students, postdoctoral fellows, and research associates) will be trained to independently use the Pulse EPR, and instrument time will be determined using the existing scheduling system for departmental instrumentation. A local advisory committee will provide oversight of the Pulse EPR and its operations. The high-sensitivity Pulse EPR will be an integral component of the biophysical instrumentation portfolio at Saint Louis University and will have a significant impact on the NIH-funded research in the region.

NSF Awards · FY 2024 · 2024-09

Understanding how people move and respond during natural disasters is crucial for improving disaster response strategies and building resilient communities. This project seeks to address gaps in current research by developing a comprehensive framework that uses advanced algorithms and large-scale data from anonymized mobile phones to model human behavior and movement during disasters. This innovative approach will identify potential vulnerabilities and forecast areas at greater risk, thereby enhancing disaster preparedness and response. The project aims to introduce three novel algorithms to better understand the interplay between human mobility and socioeconomic factors. Doing so will provide a detailed representation of how individuals and communities react to catastrophic events. This work supports the national interest by promoting scientific progress, advancing public health and welfare, and enhancing national security. The broader impacts of this project include guiding the creation of more effective and equitable disaster preparedness and response strategies, which are accessible to all community members, particularly vulnerable groups. The project aims to advance the understanding of human mobility patterns under natural hazard impacts by developing and integrating three novel algorithms: a multidimensional Dynamic-Time-Warping Self-Organizing Map (mDTW-SOM) for clustering human mobility trajectories, a Weighted Greedy Gaussian Multivariate Segmentation (WGGS) for characterizing aggregated human mobility patterns at the political geographic unit level, and a Multinomial Geographically Weighted Elastic Net (M-GWEN) for selecting socioeconomic impactors under conditions of spatial non-stationarity. These algorithms will analyze large-scale spatiotemporal datasets to capture the intricate relationship between human mobility and socioeconomic factors, providing an in-depth analysis of human behavior during disasters. The project’s methodological innovations significantly advance disaster response analysis, offering crucial insights for enhancing disaster response strategies and developing predictive models of human behavior in crises. The findings will significantly contribute to the understanding of disaster resilience and response mechanisms, facilitating more refined analyses of population behaviors and movements during disasters. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Volumetric muscle loss (VML) is a debilitating condition that leads to chronic functional impairment due to the irrecoverable loss of muscle tissue. There are no physical rehabilitation or surgical standards of care for VML. As such, there is a critical need to develop therapeutics for muscle regeneration. Animal model studies have shown that increased mechanical loading of muscle via electrical stimulation or wheel running is a promising strategy for partially restoring function. Specifically, electrically stimulated eccentric contraction (ESEC) training where the muscle-tendon unit is elongated while the muscle is contracted has been most promising. Regardless of the therapeutic approach to treating VML, and even more broadly to muscle function in general, there are no techniques for noninvasively and directly measuring the in vivo force generating capacity of muscle in animals. Shear wave tensiometry has recently been introduced as a noninvasive technique for assessing in vivo muscle-tendon loading in humans. Loading is quantified by tracking the vibrational behavior of propagating shear waves. While this technology has proven useful in assessing muscle-tendon loading across a range of human health conditions, it has not been scaled or evaluated in animal models, although it holds substantial potential in this context. Accordingly, our overall objectives are to (1) develop a small animal tensiometer capable of measuring reductions in in vivo muscle forces due to injury, and (2) determine the effectiveness of ESEC on improving muscle force generation following injury. In Aim 1, we will develop a shear wave tensiometer that integrates with our established rodent ankle dynamometer. Following VML to the lateral gastrocnemius in a rodent model, wave speed via tensiometry and ankle torque via dynamometry in injured animals will be compared to controls throughout recovery. This will mark a pivotal first step in validating the effectiveness, accuracy, and reliability of tensiometry against established methodologies in the field. In Aim 2, using the same VML model, ESEC will be applied after injury to assess its effectiveness on improving functional and histological outcomes. Successful completion of these aims holds dual significance. First, we will have validated the use of tensiometry as a noninvasive, direct measure of in vivo muscle function in small animal models. Long-term, we envision this technology as a wearable sensor to characterize muscle loading in a wide range of animal models of disease, injury, development, and aging. Second, we will show that ESEC training has the potential to make significant improvements in muscle function following VML. This will set the stage for investigating synergist treatment strategies for VML when combined with complementary therapeutics such as biomaterials or drugs.

NSF Awards · FY 2024 · 2024-09

Spaces are often better understood by cutting them into pieces of lower dimension. For example, on a topographical map of a mountainous area, the 1-dimensional lines of constant altitude provide valuable information about a landscape. The breaks in the smooth pattern of contour lines indicate significant locations---high points, low points, and mountain passes. Moreover, the lines tell us how water will flow over the landscape. The pattern formed by these lines is known in mathematics as a "foliation," and the paths taken by water determine a "flow." This project focuses on flows associated to foliations, but in one dimension higher than the map example: the foliations are comprised of 2-dimensional pieces inside a 3-dimensional space, similar to the universe in which we live. The PI will address longstanding open questions relating the fields of geometry, topology, and dynamics in 3-dimensions. In addition to original research, this project will involve the training of graduate students and support a mathematics periodical run by undergraduates and faculty at the PI's institution. The project's overarching goal is to better understand the relationship between taut foliations and pseudo-Anosov flows in 3-manifolds, and to leverage this relationship to better understand the structure of the Thurston norm. A first concrete goal is to prove, with C.C. Tsang, a strengthening of a famous unpublished theorem of Gabai and Mosher: every taut finite depth foliation of a compact, irreducible, atoroidal 3-manifold is almost transverse to a pseudo-Anosov flow. The strengthening consists of characterizing when the construction yields a pseudo-Anosov flow “without perfect fits,” a property relevant to the study of the Thurston norm. A second goal builds on the first, and is to relate the collection of pseudo-Anosov flows almost transverse to a given taut finite depth foliation to the collection of universal circles for that foliation. The PI aims to link these two families using the Gabai-Mosher construction, giving an approach to Mosher's Transverse Finiteness Conjecture. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-08

Project Summary Aging is a major risk factor for many chronic diseases including diabetes, cardiovascular disease, cancer, and neurodegenerative diseases, as well as for geriatric syndromes including frailty and sarcopenia. Therapeutics that delay and/or slow the progression of aging, and thereby increase healthspan and longevity, have remained elusive. Over the past several decades, genetic studies in model organisms and humans have identified and validated a number of longevity-associated genes. A notable subset of these genes function in the somatotropic axis that signals nutrient status and thereby regulates growth and metabolism. Together, these findings strongly suggest that modest changes in the expression levels and/or activities of these genes can have significant impact on healthspan and lifespan. Despite this strong genetic basis, how to pharmacologically modulate levels of these genes in vivo has been a major challenge. Fortunately, antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality, and they enable highly specific modulation of target protein levels in vivo. Leveraging recent advances in ASO technology, we are now well poised to target longevity-associated genes in vivo. In this project, we will: 1) design and test ASOs to modulate the levels of four longevity-associated genes, and 2) test if the ASOs can extend lifespan and improve healthspan using a mouse model of accelerated aging. Completion of these studies will provide important insights into the utility of ASO-based therapeutic approaches for modulating levels of longevity- associated genes to improve healthspan and increase longevity.

NIH Research Projects · FY 2024 · 2024-08

There is an urgent need for a better understanding of the propagation of encephalitogenic autoimmune responses and the identification of relevant immune cells and their specific characteristics, which could serve as biomarkers and therapeutical targets. CD4+ T cells show critical pathogenic roles in multiple sclerosis (MS) in preclinical models and human disease. Within the central nervous system (CNS), the encephalitogenic autoimmune CD4+ T cells infiltrate the MS brain lesions, and these T cells recirculate to the peripheral immune system, where they can be maintained and activated. The mechanisms that support long-term maintenance of the pro-encephalitogenic autoimmune CD4+ T cells remain a mystery. The long-lived stem-like CD4+ T cells (CD4+ TSTEMS) with enhanced capacities for effector differentiation were proposed to contribute to the onset and exacerbation of MS disease in humans and in the corresponding animal models. Unlike the well-characterized CD8+ TSTEMS involved in anti-tumor and anti-viral immune responses, these MS-related autoimmune CD4+ TSTEMS are still understudied. Moreover, the current markers of CD4+ TSTEMS are contextual, e.g., T cell factor 1 (Tcf1, encoded by Tcf7) is expressed in non-stem cells, including naive T cells. A lack of precision analytical tools and clearly defined markers hinders further progress in understanding the stem-like paradigm. Our published results uncovered that pro-encephalitogenic CD4+ pre-effectors de novo induced from naive T cells become programmed for diverse autoimmune TEFFS differentiation in an MS model, lending support to the stem-like paradigm. We developed a strategy to overcome the lack of biomarkers issue by identifying a consensus signature from the transcriptomes of various bona fide stem cells. We used this consensus signature and identified a conserved stemness transcriptomic profile specifically in mouse pre-effectors and human T cells from the peripheral blood of MS patients but not in MS-free controls. Furthermore, we have developed a new single- cell sequencing analytical approach enabled by an advanced scoring method, which retrieves biologically relevant information based on the cumulative expression of multiple genes involved in specific biological processes. We extend these exciting research endeavors in this proposal and dedicate Aims 1 and 2 to rigorously test the hypothesis that a continuing stemness and encephalitogenic TEFFS differentiation results in a functional and transcriptomic diversification of CD4+ TSTEM lineages under evolving autoimmune conditions. Furthermore, we have also found increased expression of the homeodomain-only protein (Hopx), in pro-encephalitogenic pre- effectors. Mouse Hopx and human HOPX are well-established orchestrators of stemness in progenitor populations. Intriguingly, we found that Hopx also critically enhanced the survival of Foxp3+ TREGS, suppressing disease in MS models. Therefore, in Aim 3, we will test the hypothesis that Hopx orchestrates independent mechanisms in pro-encephalitogenic TSTEMS and anti-encephalitogenic TREGS, resulting in functionally opposing outcomes impacting the autoimmune response.

NSF Awards · FY 2024 · 2024-08

Oxygen is essential for all animals to thrive. Within the natural world, there is significant variation between different species’ ability to temporarily live in its complete absence, a condition that is also called anoxia. Most mammals, including humans, can survive for several minutes of anoxia before tissue injury accumulates. The most vulnerable tissues are those with high rates of metabolism, especially the heart and brain. Indeed, brain injury caused by the cessation of oxygen delivery because of cardiac arrest or stroke is a major human health and societal problem. Many aquatic animals are also vulnerable to periods of anoxia because of human-induced releases of water or chemicals that can cause oxygen depletion. Pond turtles, especially painted turtles, show an extreme tolerance to anoxia, surviving for many months while overwintering in ice-locked ponds. This research aims to understand how the brain of anoxia-tolerant turtles function without anoxia, with the overarching goals of understanding the basic requirements for neurological anoxia tolerance, and to identify novel therapeutic strategies to minimize cellular injury in anoxia-sensitive species, including humans. The work includes studies of anoxia and temperature on the function of isolated brain cells and sensory function and on gene expression in different regions of the turtle brain. It will enhance the STEM workforce by training a postdoctoral fellow, a graduate student and multiple undergraduate students. It will also create a STEM learning experience for middle school children from economically disadvantaged backgrounds within the St. Louis metropolitan area. The work will exploit a recent breakthrough isolating and culturing primary cortical neurons from painted turtles to test hypotheses concerning the control of ion and synaptic arrest, with the ultimate goal of determining if the anoxia-tolerant functional phenotype is intrinsic to isolated turtle neurons or requires extrinsic factors, such as low pH, lactate, GABA, and adenosine. The effects of these factors on NMDA and AMPA receptor and voltage-gated sodium channel function under normoxic and severely hypoxic conditions using intracellular calcium recordings and whole-cell voltage clamping will also be studied. It will investigate how complex sensory functions formed by neuronal circuits are affected by anoxia and temperature in the visual and auditory systems using reduced brain preparations. It was previously discovered that evoked potentials in the brainstem during displacement of the tympanum are unaffected by severe hypoxia in turtles acclimated to 20oC, indicating that turtles may still hear while anoxic. This differs from optic tectum and retina, which show suppression. These patterns will be studied under more ecologically relevant conditions, i.e., when normoxic or severely hypoxic while acclimated to 3oC. Finally, the project will use next-generation sequencing to understand spatial gene expression patterns in turtle brain, particularly in cerebrocortex, midbrain, and brainstem with cold-acclimation and anoxia. The work should reveal factors responsible for the adaptive functional responses of single turtle neurons to anoxia, how sensory systems comprised of complex neural circuits with many neurons respond to anoxia and cold-acclimation, and the molecular signatures that underlie these functional responses. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

The lymphatic system plays a crucial role in keeping tissues in the body balanced by filtering and absorbing fluids, monitoring which cells enter a tissue, and transporting dietary fats into the bloodstream. Sometimes, vessels in the lymphatic system can become leaky. Though helpful in certain conditions, when this leakiness continues for too long, it can slow down the flow of lymph fluid, leading to excess fat buildup, tissue scarring, and persistent inflammation, which are common in many chronic diseases. This project aims to use both mathematical modeling and experiments to understand what regulates leakiness in the lymphatic system. Results from this project will ultimately guide approaches to control the lymphatic system. To broaden the impact of this project to society, high school students will participate in 10-week summer research projects that will enrich their educational training and provide a path to contribute to scientific research. The goal of this project is to develop an integrated computational and experimental engineering-based approach to determine strategies to modulate lymphatic permeability. Using a systems biology approach that iterates between experiments and modeling, the PIs will develop the first models that determine how lipids regulate CD36 (a long-chain fatty acid (FA) translocase) turnover, lymphatic vessel integrity, and cell signaling. Mechanistic differential equation modeling and data-driven regression modeling will be used to simulate the effects of CD36 across scales. Biotinylated surface assays, immunofluorescence microscopy, direct measurements of LEC permeability, and morphological assessment of LEC junctions will be used to inform and calibrate the models. The experimentally validated models will be applied to predict strategies to modulate lymphatic permeability. More broadly, the mechanistic, quantitative, and multiscale understanding of how CD36 regulates lymphatic permeability produced in this project represents significant advances in lymphatic biology and bioengineering that can be leveraged to address pathological changes in several diseases, e.g., obesity and the metabolic syndrome. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY. A great accomplishment of modern medicine is the increased lifespan of the population worldwide. Unfortunately, longevity also increased the healthcare burden due to comorbidities of aging such as atherosclerotic cardio- vascular disease (CVD). Overwhelming evidence indicates that hyperlipidemia is a main driver of CVD, which promotes an inflammatory response by vasculature-resident macrophages. Other inflammatory cells, chiefly among them T-cells, are also recruited to growing plaques and play a fundamental role in their remodeling and susceptibility to rupture. In women, it is well recognized that loss of ovarian hormones at menopause is an independent risk factor for cardiovascular events. Current therapies, such as statins, lower circulating LDL- cholesterol and have been clinically demonstrated to decrease mortality in the elderly. Yet, despite the use of statins for over five decades, atherosclerosis still remains the leading cause of death in men and women globally. These observations indicate that 1) there are additional, yet unidentified, lipid-independent mechanisms that contribute to atheroprogression and plaque rupture; and 2) additional therapies are needed to treat ASCVD. Unpublished preliminary data from our laboratories in mice show that aging in both sexes and loss of estrogen in females promotes T-cell mediated inflammation. We hypothesize that this age- and menopause-dependent inflammation exacerbates underlying CVD such that further lowering cholesterol does not provide added benefit. Here we will test the idea that promoting an anti-inflammatory, pro-resolving environment in the plaque is atheroprotective. To accomplish this, we will use treat mice with pulsed low-dose RANKL (pRL), which we show induces plaque resident regulatory CD8+FoxP3+CD25+CTLA4+ T-cells that secrete IL-10. If successful, pRL will be a novel therapeutic for treating ASCVD, that works by mechanism distinct from therapies currently in use. Additionally, the proposed studies will deliver two key innovations. First, we will compare atheromata in young (2-month-old) and old (18-month-old at beginning of intervention, corresponding to ~60-year-old human) male and female mice. Second, old females will be ovariectomized to mimic human menopause. Thus, in females we will segregate the effects of aging and menopause on plaque development. We will comprehensively map athero-prone proximal aorta transcriptomic profiles at a single-cell resolution, focusing on inflammatory cell clusters, cell-to-cell communication networks, T-cell-mediated dysfunction of smooth muscle cells and endothelial cells, markers linked to plaque stability/vulnerability, and transcripts encoding predicted secreted proteins (secretome). The integrated mechanistic data obtained with our unprecedented discovery approach will have a lasting impact in the field of vascular physiology and pathology because most published murine atherosclerosis studies have systematically ignored the effects of age, sex, and hormonal status, despite most CVD patients being older (and post-menopausal). Overall, these outcomes will be the foundation for future mechanistic studies and may identify novel plaque-secreted biomarkers to assess disease severity.

NIH Research Projects · FY 2024 · 2024-08

ABSTRACT. The G-protein Gb3 subunit encoded by the gene GNB3, is the protein present in multiple tissues and cell types, including retinal cone photoreceptors, as part of the Gabg heterotrimers responsible for the intracellular signaling initiated by the G-protein-coupled receptors, GPCRs. There is evidence that mutations in GNB3 are associated with cardiovascular disease, metabolic syndrome, obesity and visual impairment. Rare GNB3 mutations have been found associated with retinal degeneration and congenital stationary night blindness. The mechanisms of these pathophysiological conditions linked to GNB3 are poorly understood. In the retina, there is little understanding of why members of the same gene family GNB3 and GNB1 are expressed selectively in cone and rod photoreceptors, and what roles Gb3 and Gb1, in tight complexes with the corresponding Gg subunit, Ggc and Gg1, contribute to the distinct properties of cone and rod phototransduction and retinal diseases. This pilot proposal focuses on the initial characterization of a unique mouse model that our laboratory has developed to replace the entire rod Gb1g1 subunit complex with its cone analogue Gb3gc. The proposal aims to collect critical preliminary data for a more comprehensive project focusing on the specificity of retinal signaling and mechanisms of retinal disorders involving Gnb3. This project builds the necessary foundation for further mechanistic studies of specific human mutations, as well as the development of new therapeutic approaches.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY/ABSTRACT Ion channels in the voltage-gated ion channel (VGIC) superfamily play pivotal roles in virtually all physiological processes. This proposal delves into essential constituents of this superfamily, focusing on elucidating the mechanisms governing voltage sensing and electromechanical coupling of these proteins in pathophysiological contexts. Our investigation focuses on discerning the regulatory interplay between the voltage-sensing domain (VSD) of VGICs and the lipid-ordered membrane domain (OMD) enriched with cholesterol. Recent findings from our laboratory have unraveled the significant influence of OMD on modulating membrane excitability in somatosensory dorsal root ganglion (DRG) neurons. A reduction in OMD dimensions, leading to augmented native ionic currents of pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, emerges as a contributing factor in neuropathic pain and inflammatory pain. These distinct lipid nanodomains exercise direct control over the voltage sensor of HCN channels, consequently influencing channel opening. This aspect of HCN channel regulation, integral to neuronal excitability and cardiac pacemaking, necessitates in-depth investigation. Our biophysical approach employs patch-clamp fluorometry (PCF) combined with fluorescence lifetime imaging microscopy (FLIM) to measure voltage-sensor conformational dynamics within native lipid environments. Leveraging genetic-code expansion featuring noncanonical amino acids and bio- orthogonal fluorescence labeling through click chemistry enables site-specific labeling and facilitates Förster resonance energy transfer (FRET) measurements. Furthermore, our approach includes an improved transition metal FRET (tmFRET) technique in conjunction with phasor plot FLIM, with the primary goal of investigating the potential intermediate states of VGIC voltage sensors. By successfully implementing this approach, we intend to discern the influence of OMD localization, lipid composition, and disease-associated mutations on these intermediate voltage sensor states. This extensive investigation holds the promise of significantly deepening our understanding of the voltage-sensing mechanism of VGIC in physiological processes.

NSF Awards · FY 2024 · 2024-07

The 32nd IEEE International Conference on Network Protocols (ICNP) will take place in Charleroi, Belgium, from October 28-31, 2024. This project supports the participation of 15-20 US-based graduate students in this conference. Participation in conferences like ICNP is essential for graduate students, allowing them to engage with leading researchers and gain exposure to the latest advancements in network protocols. This experience contributes to their education and professional development, aligning with the National Science Foundation's mission. By attending ICNP, students will have the opportunity to present their research, receive feedback, and network with professionals from around the world. This interaction fosters the exchange of ideas and innovation, advancing the field of network protocols. The goals of the project are to enhance the educational experience of graduate students, promote diversity and idea exchange in the field of network protocols, and foster international collaborations. The methods include providing travel grants to students selected by the travel grant committee, organizing workshops and social networking events, and ensuring the inclusion of diverse participants. By attending ICNP, students will gain valuable insights into the latest research and developments in network protocols, contributing to their academic and professional growth. The ICNP conference has established itself as a premier international event, providing a unique opportunity for students to expand their knowledge, enhance their research skills, and gain a broader technical perspective. This experience is anticipated to significantly impact the students' academic and professional development, justifying the expenditure of federal funds to support their participation. This experience is anticipated to have a significant positive impact on the students' academic and professional development. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2024 · 2024-06

Project Abstract: Retrovirus intasomes are integrase (IN) multimers bound to two viral DNA ends that are capable of concerted integration into a target DNA. Assembly mechanisms are unknown for IN multimers in different retrovirus intasomes, whose structures contain four IN subunits (prototype foamy virus, delta retroviruses), or 4 to 8 (alpha and beta retroviruses) or 4 to 12 and up to 16 subunits (lentiviruses). The 3-dimensional structure of different retrovirus intasomes has revealed remarkable architectural diversities besides conserved structural features and catalytic mechanisms. To understand the assembly mechanisms, we have determined 1) biophysically that the Rous sarcoma virus (RSV) tetrameric intasome is the precursor to the mature octameric intasome with four IN dimers; 2) intasome assembly in vitro is controlled by the C-terminal “tail” region comprising the last 17 residues of IN; and 3) the structure of RSV octameric intasome (containing four IN dimers and two viral DNA) and strand transfer complex (STC) with four IN dimers and a viral/target DNA substrate. We will determine the structure of RSV tetrameric intasome, a novel intermediate in RSV integration pathway by cryo-EM. We recently determined the structure of RSV octameric intasome by single particle cryo-EM at an overall resolution of 3.2 Å. The conserved intasome core (CIC) that contains the catalytic center for concerted integration had a resolution of 2.8 Å. The ensemble of conformations in the intasomes revealed significant dynamic flexibility of the two non-catalytic distal IN dimers, along with some movement of the two catalytic proximal dimers contained in the CIC, previously unrecognized in retrovirus intasomes. We hypothesize that there are ordered conformational transitions between key intermediates in the assembly pathway of RSV intasomes and the subsequent capture of the host target DNA. Our proposed studies will reveal how RSV IN first assembles the precursor tetrameric intasome en route to the mature octameric intasome by the following Aims. Aim 1. Determine the structure of the RSV intasomes. We will determine the structure of the RSV tetrameric intasome by cryo-EM. We will determine the first structure of intasome assembled with both U3 and U5 LTRs to provide novel insights into the assembly of intasomes most similar to biological complexes. Aim 2. Determine the functional significances of tetrameric and octameric intasomes. We will use structural and virological approaches to increase our knowledge of retrovirus integration, enhanced by near- atomic structure-based missense mutagenesis of RSV IN. We will focus on the transition of the tetrameric intasome to the octameric form to probe the independent functions of the distal and proximal IN dimers. Virological effects of single-point mutations in IN will be measured by their effect on reverse transcription and integration in virus infected cells and electron microscopy of virions to define phenotype under physiological conditions. These projects will expand our understanding of intasomes functional states and determine how higher order structures influence integration, and hence retroviral replication.

NIH Research Projects · FY 2026 · 2024-05

Intrinsically disordered proteins (IDPs) lack a stable globular structure and are prevalent in the human proteome. They have many important biological functions, including serving as scaffold platforms that coordinate multiple proteins, forming membrane-less organelles, and acting as protein and nucleic acid chaperones. We discovered an unstructured protein domain within the PALB2 protein (Partner and Localizer of BRCA2) that recombines nucleic acid strands. Homologous recombination (HR) is a complex multistep reaction essential for all living organisms and directed by only a few known globular proteins. Recombination supported by the intrinsically disordered region (IDR) of PALB2 represents a novel function of IDPs and a new paradigm in nucleic acid metabolism. This proposal will establish the molecular mechanism of this novel IDP function. Sequence and structural properties of the PALB2 DNA-binding domain (DBD) and enrichment of eukaryotic DNA repair factors with disordered DBDs suggest a general hypothesis that similar regions evolved as a common tool to handle complex multistrand DNA and RNA intermediates during chromosome repair. We will investigate this hypothesis using the PALB2-binding domain of BRCA1. Results will be critical for establishing a functional role the PALB2-DBD IDR in DNA repair and will help to identify similar IDRs in other DNA and RNA metabolism factors. PALB2 is a central hub for large DNA repair complexes that mediates HR or a homology-directed repair (HDR) of chromosome breaks. PALB2 interacts with at least a dozen proteins, including breast cancer susceptibility proteins 1 and 2 (BRCA1, BRCA2) and the major homologous recombinase RAD51. We identified major DNA-binding amino acids in the PALB2-DBD that significantly contribute to DNA repair in cells. We discovered that PALB2-DBD is structurally disordered and can direct DNA or RNA strand exchange. We demonstrated that PALB2-DBD forms a compact dimer and condenses ssDNA. Thus, we hypothesize that a novel chaperone-like mechanism promotes PALB2-DBD-mediated strand exchange. Here, we will investigate this hypothesis using a set of complementary structural and spectroscopy methods (including single-molecule analysis) to determine conformational changes of PALB2-DBD and DNA. Furthermore, we will generate separation-of-function mutants for future studies of the physiological role of PALB2-mediated strand exchange in cells and will determine how the disordered PALB2-DBD regulates the activity of major recombinase RAD51. This work will establish the structural and mechanistic bases of PALB2 recombinational properties, will provide insights into the function of PALB2-mediated strand exchange in cellular DNA repair and genome maintenance, and will drive research of other scaffold proteins, such as BRCA1 and BRCA2, with intrinsically disordered DBDs. These results will yield unique insights into cancer etiology, aging, neurodegeneration, and other pathologies.

NIH Research Projects · FY 2025 · 2024-04

Project summary This project aims to investigate a novel approach to mitigate the risk of vascular thromboses, which imposes a significant economic burden in the US. Our strategy is to develop a technology that, by specifically targeting the lipid-binding domains of factor X (read factor ten) and prothrombin, two essential coagulation factors, regulates the rate at which factor Xa and thrombin are produced and, consequently, controls the formation of blood clots. This approach is unique compared to current pharmacologic methods that either directly (DOACs) or indirectly (heparins) target the active site of clotting proteases or impair the proper synthesis of a family of proteins to which multiple coagulation factors belong (warfarin). We propose this approach will offer potential benefits for patients with prothrombotic antiphospholipid antibodies who experience limited effectiveness with existing medications. Antiphospholipid antibodies are a defining feature of antiphospholipid syndrome (APS), which is an acquired autoimmune disorder. Additionally, there is evidence of their involvement in infectious diseases, including COVID-19. By using an in vitro selection process, our preliminary studies identified a nanobody suitable for testing this hypothesis, which we called NanoProTEN. Aim 1 of this project aims to investigate the biochemical characteristics of NanoProTEN, including its affinity and specificity towards coagulation factors and plasma proteins involved in the blood clotting cascade. We will then assess its anticoagulant potential in human plasma and with purified coagulation factors, and its ability to interfere with antiphospholipid antibodies. These studies will define the molecular interactions of NanoProTEN, evaluate its potential off-target effects, and establish its anticoagulant potential compared to existing therapies. Studies in Aim 2 will define the unique mechanism of action of NanoProTEN through structural studies using cryo-electron microscopy. By determining the structures of NanoProTEN bound to prothrombin and factor X, we will uncover the structural elements contributing to its dual selectivity. These studies will determine how NanoProTEN interacts with these factors, offering mechanistic insights into its mode of action and providing essential information for enhancing its activity. In conclusion, the primary outcome of this research project is the development of an innovative technology represented by NanoProTEN. This technology holds promise as a versatile research tool and a potential candidate for a new class of anticoagulants for improving outcomes in patients with prothrombotic autoantibodies and potentially other thrombotic disorders.

NIH Research Projects · FY 2025 · 2024-03

PROJECT SUMMARY Neuropathic pain is a pervasive and debilitating condition associated with modifications in synaptic plasticity and altered gene expression within the spinal cord. However, the specific molecular mechanisms underlying these synaptic changes remain poorly understood. This proposed project aims to unravel the synaptic transcriptome modifications occurring during neuropathic pain and investigate the involvement of G- quadruplexes in translocation of transcripts at the synapse. The outcomes of this study will provide a comprehensive map of the synaptic transcriptomic changes in neuropathic pain and elucidate some of the factors contributing to this alteration. Furthermore, it will pave the way for the development of innovative therapeutic strategies targeting the G-quadruplexes, ultimately offering new avenues for the management of neuropathic pain.

NIH Research Projects · FY 2026 · 2024-02

Project Summary Carbohydrates form the basis of all living organisms and are ubiquitous both in nature and in medicine. However, methods for the chemical synthesis of carbohydrates remain cumbersome. The rapid growth of R&D in glycoscience demands the development of rapid, efficient, and simple procedures for glycan synthesis. This proposal seeks to meet this demand by focusing on the development of an affordable and accessible automation platform that will enable both specialists and non-specialists to perform the synthesis of glycans from renewable precursors. Current methods for the synthesis of glycans are highly sophisticated and operationally complex. By contrast, high performance liquid chromatography equipment-based automation (HPLC-A) represents a highly accessible method for synthesis because many scientists already have easy access to HPLC equipment. Automated synthesis offers operational simplicity by delivering all reagents using standard HPLC components, but also convenient real-time reaction monitoring of every step. Some of our automated reactions have been accurately reproduced by a minimally trained high-school student researcher. Unexpected recent pandemic revealed unpreparedness of our society, but also gave us an opportunity to showcase how HPLC-A can be utilized to enhance our productivity. This proposal aims to improve our HPLC-A setup by introducing a universal platform for completely automated synthesis of glycans from simple and renewable precursors. We propose to develop a dedicated automated circuit for the synthesis of sugar building blocks and will develop dedicated glycosylation methods that will connect monosaccharides into oligomeric networks in the automated setting modes for syntheses on solid supports and in solution. We will accessorize our system with new modules and attachments to achieve high efficiency of synthetic steps, improve reproducibility of reactions, help to reduce reagent and solvent excess. We will then demonstrate how well these strategic adjustments work in the completely automated, “press of a button” production of glycans. We are currently interested in glycans found in human milk, but the developed methods and technologies will also allow to achieve other sequences. Upon completion of the proposed studies, we expect to have achieved a reliable and simple platform for completely automated synthesis of glycans from simple and renewable precursors. Investigators with access to standard HPLC equipment should be able to perform automated synthesis using our methods. Machine- assisted synthesis ensures rigorous experimental design to obtain robust results, to eliminate variability, and to accurately reproduce experiments multiple times by different users. Synthesis of glycans using this user- friendly, automated platform will accelerate discovery in many scientific disciplines and can significantly impact technology, society, the economy, and public health.