Grants

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Implementation of the RTS,S malaria vaccine in sub Saharan Africa has been recommended by the WHO and UNICEF has pledged $170 million for production of the first vaccine supply. Because the efficacy of RTS,S wanes within one year from the primary doses, the regimen includes a 4th booster dose. However, based on findings from the phase 3 trial, the magnitude of Ab responses induced by the booster dose is significantly lower than those induced by the primary regimen. The booster dose increased duration of efficacy marginally. The regimen of another leading vaccine candidate, R21, also requires a booster dose. Understanding the immune mechanisms underlying poor booster dose responses will be important to support the design of future implementation studies and optimize actual implementation of RTS,S and other malaria vaccines. The limited effect of the booster dose might be explained by repeated exposure to malaria compounded to exposure to other infections after the first months of life. Extensive epidemiological evidence supports this hypothesis including a recent trial that showed that efficacy of administration of RTS,S in combination with repeated chemoprophylaxis was higher than RTS,S or chemoprophylaxis alone. Repeated malaria exposure may negatively affect Ab magnitude and functional activity via effects on innate, memory cells, and T cells. Additionally, our data, and data from others suggest that deficiency of micronutrients, particularly iron and zinc, acquired after cessation of breast feeding may impact responses to the booster dose. A third determinant of reduced responses to the booster dose could be the suboptimal spacing between the primary and booster doses, whereby memory B cells induced by the primary doses have waned before the booster dose. Following a cohort of 600 Malawian children from the primary vaccination until eight months after the booster dose, we will undertake comprehensive immunologic analyses to investigate the mechanisms underpinning the poor responses to the booster. We will identify the specific Ab responses and functions that are suboptimal after the booster dose. Additionally, we will i) quantify innate cell responses with and without the stimulation with AS01, and assess epigenetic changes leading to tolerogenic responses in monocytes; ii) quantify subpopulations of vaccine specific CD4+ T follicular helper cells and regulatory cells and their effect on Ab and B cell responses; iii) quantify induction and duration of memory cells, that may be impairing Ab responses to the booster dose; iv) assess whether exposure to malaria parasites, and micronutrient deficiency may be affecting all these immune responses; v) evaluate whether children with dysfunctional immunity to RTS,S also have impaired immunity to other childhood vaccines associated with the same determinants. Data generated by this study will help elucidate the basis of the limited effect of the booster dose and, thus, will be crucial to inform strategies to optimize boosting of the RTS,S-induced response.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY By mentoring junior investigators from the United States and low-come countries (LICs) in patient- oriented research (POR) in global health, particularly in vaccine-preventable diseases, I hope to build local capacity, improve outcomes and reduce health disparities. Working within the resource-limited health systems in LICs is challenging, and the influences of co-infections (particularly parasitic infections), anemia, undernutrition, and various microorganisms on vaccine efficacy may be underappreciated. For these reasons, vaccines and vaccine campaigns developed for and assessed in high-income countries (HICs) cannot be assumed to be feasibly implemented or to perform similarly in LICs. For instance, SARS-CoV-2 vaccination with mRNA-based vaccines in sub-Saharan Africa has been limited by the cold-chain requirement of these vaccines. Cultural and socio-economic factors have led Malawians to generally choose receiving the single dose Ad26.COV2-S rather than the ChAdOx1 vaccine. Growing evidence suggests that performance of vaccines in LICs is lower than HICs and the protective immunity elicited by some vaccines is shorter than originally anticipated. In an interconnected world where diseases do not have borders, optimizing interventions to control transmittable diseases in LICs impacts the health of populations worldwide. The overall goal is to train junior investigators across disciplines to optimize vaccinations in Malawi, a sub-Saharan African country in which I have established a thriving research infrastructure. The first two aims of this application, supported by my R01AI164686-funded project, will assess i) the longevity of antibodies (magnitude and function) and memory cells induced by the Ad26.COV2-S SARS-CoV-2 vaccine; ii) the contribution of pre-existing changes in innate immunity to these responses; iii) the association of malaria, micronutrient deficiency, and microorganisms colonizing the nasopharynx with these responses. In the third aim, funded through this proposal, we will address the longevity of five childhood vaccines, focusing particularly on optimizing the timing of re-vaccination. The importance of this question became obvious in 2023 when three poliovirus cases were identified in Malawi. The Ministry of Health, with no relevant data available, decided to revaccinate all children with an arbitrary cutoff of 15 years of age. I have mentored several investigators in POR, particularly from Malawi, and have been conducting research in the immunoepidemiology of vaccines in LICs (including Malawi) since 2015. With this award, I would be able to expand my panel of trainees, devote more time to each of them, acquire training to further my mentorship skills as well as my own professional skillset, and expand the focus of the research to include other childhood vaccines. With the research infrastructure built in Malawi, I am now well-equipped to successfully train clinician scientists and other junior investigators in POR in infectious diseases, particularly in vaccines in LICS.

NIAID - National Institute of Allergy and Infectious Diseases

Summary Ebola virus (EBOV), a filovirus, causes periodic outbreaks with high fatality rates. To understand the basis for viral pathogenesis, emergence, and to devise control strategies, it is important to define the mechanisms that underlie EBOV assembly and release of infectious virus. The VP24 protein, produced by one of seven viral genes, is a 24 kDa multifunctional protein unique to the filovirus family. EBOV VP24 blocks cellular responses to interferons (IFN) by preventing nuclear translocation of tyrosine phosphorylated STAT1 (pSTAT1), a transcription factor central to antiviral IFN signaling. VP24 accomplishes this by interacting with importin α (IMPA) nuclear transport proteins, preventing binding and nuclear import of STAT1, a key transcription factor needed for IFN responses. That VP24 interacts with IMPA nuclear import factors suggests that VP24 can traffic into the nucleus. In addition, transfection studies identified a nuclear export signal (NES) at the C-terminus of EBOV VP24, further supporting nuclear its trafficking. However, whether EBOV VP24 undergoes nucleocytoplasmic trafficking and the functional significance of this trafficking remain to be determined. VP24 also plays a critical but incompletely understood role in viral genome packaging and production of infectious viral particles. Most notably, VP24, the EBOV nucleoprotein and EBOV VP35 together form filamentous nucleocapsid structures, called nucleocapsid- like structures (NCLS) that are morphologically indistinguishable from the nucleocapsids present in EBOV virions. The presence of VP24 also renders NCLS competent for actin-dependent transport. Fundamental questions remain including how VP24 facilitates actin-dependent transport and the specific molecular features required for NCLS formation and trafficking. Our Preliminary Data indicates that VP24 can traffic into and out of the nucleus and that disruption of IMPA binding significantly impairs virus growth at a late stage in the replication cycle. Disruption of nuclear export function completely abrogates infectious particle production. Consistent with these observations, an NES mutant VP24 recombinant virus could not be recovered. Providing mechanistic insight, initial studies on the NES mutant VP24 suggest that it is significantly impaired for actin-dependent trafficking. Finally, proteomic analysis suggests that VP24 recruits components of the Wave regulatory complex (WRC), a regulator of Arp2/3 dependent actin polymerization via its NES sequence. These data support roles for VP24 IMPA binding and NES sequences in virus production and suggest that the NES may recruit the WRC to promote NCLS trafficking. The proposed studies will determine how VP24 NES sequences recruits the WRC to promote actin-dependent trafficking of viral nucleocapsids and incorporation into viral particles, and it will define the IFN-dependent and IFN-independent consequences of VP24-IMPA interaction.

NCI - National Cancer Institute

ABSTRACT There is an urgent need to nominate biomarkers that are likely to predict the efficacy of radiotherapy and accelerate their clinical translation. Efforts thus far have been limited in large part because the omic features regulating tumor cell survival and their frequency across and within individual cancer types had not been studied. Also, radiation is one of the most utilized therapeutic modalities in cancer. Despite this, radiotherapy dose is still delivered using a ‘one size fits most’ paradigm and has not yet lent itself to personalization based on genetic features. Our group of investigators have expertise that spans several aspects of the study of translational lung radiotherapy including advanced functional genomics, use of pre-clinical models of human tumors, personalized therapeutic interventions, bioinformatics, computational biology, and clinical research. Collectively, we have: (1) completed the largest profiling effort of the genetic vulnerability of cancer cells to irradiation to date (533 cell lines); (2) completed an arrayed unary (one mutation per well) gene variant cellular profiling project that has interrogated ~400 common and rare genetic variants for response to ionizing radiation in cells; (3) developed new functional genomic tools to study genome-phenome associations at scale; (4) developed over 500 genetically and clinically annotated patient derived xenograft (PDXs), subset of ex vivo and 3D models for testing using a radiation platform and (5), developed new computational tools and frameworks to personalize radiation dose treatments. The major themes for this proposal are: (i) analyze matched pre- and post-radiotherapy genomes from patient tissue and using PDX to elucidate mechanisms of de novo and/or acquired resistance; (ii) develop predictive diagnostics related to multi-omic tumor variates in patients receiving radiotherapy for lung cancer; (iii) integrate multi-tiered omic data to accurately predict the probability of local failures after lung radiotherapy; and (iv) identify and validate functionally significant molecular lesions in tumors that impact radiotherapeutic efficacy. This project is explicitly designed to utilize patient and model systems data in iterative and complementary testing cycles. Due to our extensive and sustained efforts in this space, we have an established preclinical understanding of the most salient genetic determinants that regulate radiation sensitivity in lung cancer. Altogether, our highly integrated proposal will come together in an unprecedented effort to advance toward clinical integration new information and capabilities that will ultimately improve outcomes for the multitude of patients receiving lung radiotherapy each year in the US and abroad.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Autoimmune type 1 diabetes (T1D) is caused by the T cell-mediated destruction of insulin producing beta (β) cells in the pancreas. The incidence of T1D is rising globally. This is thought to be linked to early life exposure to microbes, environmental pollutants, and even diet. Viral infections in mouse models of diabetes have shown both acceleration and protection of diabetes, but these differences occur at different ages and degree of insulitis. The deciding factor in whether CD4+ T cells will prime the immune system to initiate T1D is the inflammatory context of the initial peripheral antigen encounter, particularly the timing of type I interferon (IFN-I) exposure triggered by microbes. Unfortunately, the vast majority of research has utilized specific pathogen free (SPF) mouse models examining activation in the absence of IFN-I. These conditions are very different from the environment in which humans live. At UMN, we have created a ‘dirty’ mouse model or normal microbial environment (NME) to study how the immune system responds or develops in the presence of microbes and viral pathogens, a more physiological environment driven by IFN-I production. Thus, we now have a diabetes model that we can study with NME conditions. Depending on the age and duration of time in NME, our NOD mice can be completely protected from diabetes. Our goal is to understand how infections impact immunity to either trigger or protect against diabetes. A better understanding of this process could provide therapies that prevent or treat human diabetes. We will test three specific aims; 1) Determine the role of IFN-I on naïve CD4 T cell fate following TCR activation in SPF conditions, 2) Determine the mechanism(s) by which NME prevents autoimmune diabetes, and 3) Develop autoimmune diabetes therapies. Our goal is to determine the role of IFN-I during initial T cell fate decision between Teff and Tregs. We hypothesize this fate decision leads to iTregs and will focus on induction, survival or enhanced suppressive function. Finally, we will explore IFN-I therapy and engineered TCR Tregs for autoimmune therapy.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY (See instructions): Cell growth and division are the essential processes for tissue homeostasis, regeneration, and cancer development. Therefore, the study of cell growth regulation is important for understanding impaired tissue regeneration and tumorigenesis. Cell growth and cell division are coordinated via mutual regulation, and these two processes together determine a fundamental cellular property, the cell size. The molecular mechanisms underlying the linkages between these processes are largely unknown. Cell growth can trigger cell division by diluting the cell cycle inhibitor RB, which contributes to the maintenance of cell size homeostasis. The RB dilution mechanism relies on the cell cycle-dependent RB concentration dynamics, but the underlying mechanism is still unclear. On the other hand, cell cycle and cell size can feed back to regulate cell growth rate, of which the mechanisms are yet unknown. I found that the protein synthesis efficiency, a major determinant of cell growth efficiency, increases at the G1/S transition and decreases as cells grow larger within each cell cycle phase, suggesting that the global protein synthesis rate is actively regulated to facilitate cell cycle progression and cell size control. Here, in this proposal, I aim to address these gaps in our knowledge by determining the molecular mechanisms regulating RB concentration and global protein synthesis during cell cycle progression and cell growth. During the K99 phase, I’ve determined the molecular mechanisms underlying the RB concentration regulation during cell cycle progression (Aim1), which further revealed the molecular basis of how cell growth triggers cell division. During the R00 phase, I will continue to study how global protein synthesis is regulated during cell cycle progression (Aim2), which will help us understand how cell cycle regulates cell growth. These two aims link cell growth with cell division from the perspective of protein synthesis regulation. Then, the study on how protein synthesis is regulated during cell growth (Aim3), which will be pursued during R00 phase, explores the mechanism of how cell size feeds back to regulate growth rate. Thus, the three aims together set up a foundation towards a deeper understanding of the principles governing cell growth and proliferation. The completion of above aims will deepen our understanding of the fundamental molecular mechanisms regulating protein synthesis and cell growth rate, and therefore have broad impacts on cell and developmental biology. Moreover, this work will enhance our understanding of tissue regeneration deficiencies and tumorigenesis.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Monocarboxylate transporters (MCTs) mediate the transport of key metabolic intermediates, such as lactate, urate, and pyruvate, across cellular membranes. Despite their importance in metabolic homeostasis and their implication in a range of diseases—including cancer, cardiovascular disease, and neurodegeneration—the MCT family remains poorly understood. Novel approaches are needed to study the role of MCTs in physiology and disease. MCTs are highly conserved in the nematode Caenorhabditis elegans. C. elegans is a highly tractable genetic model, and its defined anatomy, simple diet, and optical transparency offers several advantages for studying conserved molecular and physiological processes. Through a genetic screen for mutants with defecation defects, I found that a point mutation in slcf-1 causes a shortened defecation cycle. slcf-1 is expressed in the intestine and regulates intestinal calcium waves (ICWs) that drive the defecation motor program (DMP). These findings implicate a novel role for MCTs, metabolism and Ca²⁺ homeostasis. slcf-1 shares homology with MCT9, which has been implicated in hyperuricemia and can contribute to cardiovascular disease, gout, and renal cell carcinoma. The overarching goal of this project is to define the role of slcf-1 in intestinal Ca2+ dynamics and metabolism. In Aim 1, I will analyze how mutations in slcf-1 affect Ca²⁺ waves. I will quantify ICW properties, such as intervals and rise and fall times, perform tissue-specific rescue, and examine SLCF-1::GFP localization. In Aim 2, I will determine SLCF-1 substrates and how SLCF-1 is dynamically regulated using HEK293T transport assays and mutant slcf-1 promotor constructs. Finally, in Aim 3, I will investigate the metabolic consequences of slcf-1 loss, assessing mitochondrial morphology, activity, and stress, alongside broader metabolic pathway alterations. These studies will provide greater insight into how conserved monocarboxylate transporters connect metabolism, calcium signaling, and intestinal motility, and promote a broader understanding of MCTs’ role in human health and disease.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Denervation injuries lead to altered neural activity in sensory processing regions of the brain. Recovery from these injuries is achieved by learning new motor skills to perform tasks of daily living; this requires successful cortical remapping. This remapping is thought to be mediated in part by changes in thalamocortical (TC) connections from the thalamus to cortex. However, the reasons that some patients recover well while others do not is poorly understood. Microglia shape circuits during development by pruning synapses in an activity dependent manner. Neurons and other cells express “eat me” and “don’t eat me” signals that act through microglial receptors to guide removal of underused synaptic connections such as those arising from the denervated body part. Large scale activity changes in sensory circuits after denervation injury lead to altered signaling through these microglial receptors, altering the activity and function of synapse-associated microglia. However, it is poorly understood how microglia coordinate with neurons to direct adult cortical circuit reorganization after peripheral injury. The goals of this project are to determine the necessity of microglia for post-injury adult circuit reorganization, determine the impact of aberrant signaling from unpruned connections, and identify the molecular mechanisms microglia and neurons utilize to coordinate their activity with other cells in the brain. To understand how microglia mediate post-denervation circuit remodeling, we will use a mouse model of unilateral infraorbital nerve transection (IONX). IONX mimics many aspects of circuit remodeling that occurs after unilateral denervation injury. We hypothesize that IONX causes microglia-mediated TC remodeling in the deprived somatosensory cortex of adult mice. This remodeling is expected to cause behavioral adaptations, particularly related to orofacial sensory/motor function. We will address this hypothesis by 1) determining the necessity of microglia for TC synapse density changes after IONX. Using tissue collected from acute, subacute and chronic timepoints, we will quantify changes in thalamocortical input density within the deprived somatosensory cortex, and microglial association with and removal of TC synapses. We will then deplete microglia to establish if microglia are required for the changes in the deprived somatosensory cortex. We will assess orofacial function in sham and IONX mice to demonstrate the impact of microglial TC circuit reorganization on behavioral adaptations. We will also modulate the activity of TC connections in the deprived somatosensory cortex to establish that preserved, erroneous activity after IONX leads to orofacial dysfunction. 2) We will use gene expression to identify the mechanisms by which microglia and neurons influence adult circuit remodeling after injury, such as identification of inactive synapses and increased phagocytosis. The findings of this proposal will improve our mechanistic understanding of how cells in the adult brain adapt to peripheral denervation injury, and can be used to gain insight into healthy adult plasticity.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Type 2 Diabetes (T2D) is estimated to affect over 30 million US adults and skeletal muscle (SkM) insulin resistance (IR) is one of the primary aberrations. SkM is a complex organ comprised of different cell types including: myofibers, endothelial cells (EC), smooth muscle cells, fibro-adipogenic progenitors, satellite cells and immune cells. Effective insulin-stimulated SkM glucose uptake is dependent on 1) insulin-stimulated activation of terminal arteriole EC to perfuse microvascular units, 2) transport of substrates across capillary ECs and 3) stimulation of myofiber insulin signaling, resulting in glucose uptake. Each of these steps can be dysregulated by pro-inflammatory cytokines released from localized immune cells in the SkM niche. Therefore, different cell types in SkM can impact insulin sensitivity. Previous research has identified a dysregulated transcriptional profile with SkM IR in the basal state and during insulin stimulation. A significant limitation of these prior studies is the restriction to whole SkM homogenates and therefore not identifying which cells or spatial area within the tissue the dysregulated signals originate from.  We have developed a pipeline for full-length amplification of single cells from SkM using the iCELL8 platform, resulting in 2-3 fold greater gene coverage than previous research, allowing us to investigate transcriptional aberrations that occur with SkM IR at a single cell resolution. Recent developments in spatial transcriptomics now permit transcriptional profiling on sectioned SkM tissue which identifies the location and proximity of dysregulated cells and how they relate to IR in SkM. This study will leverage a hyper-insulinemic euglycemic (HE) clamp with stable glucose isotope tracers and indirect calorimetry to assess insulin-stimulated glucose disposal (Rd) and non-oxidative glucose disposal (NOGD) in individuals with obesity with and without IR compared to an insulin sensitive control group. For the first time, the transcriptional responses to insulin in human SkM will be probed at a single cell and spatial resolution. We will identify which cells and areas of SkM respond to insulin and if they are spatially localized to each other and if their gene-network profiles correlate with a greater in vivo Rd and NOGD. This novel and innovative approach will reveal which cells (and importantly, where in SkM) the aberrations occur with SkM IR whilst dissociating the confounding effects of Obesity. Results from this proposal will identify specific molecular targets to alleviate SkM IR that will be targeted in future interventions.

NSF

Microbial energy and mass transfers have profound ecological and biogeochemical consequences, yet the controls directing microbial metabolic trade-offs remain poorly understood. The proposed project will study metabolic shifts in energy and mass transfers by microorganisms living through some of the earliest-evolving metabolisms known on Earth. By focusing efforts towards microbes driving primary production anaerobically with molecular hydrogen — through a metabolic process known as chemosynthesis — the project will test whether metabolic trade-offs can be predicted as a function of (i) growth temperature and/or (i) the amount of free energy associated with anaerobic respiration. State-of-the-art continuous cultivation techniques will be applied to compare changes in respiration rates, cell yields, and cell doubling times within and among chemosynthetic microbes under steady-state conditions. These experiments are key for integrating parameters describing microbial metabolic changes into mathematical and/or thermodynamic models of microbial growth and its biogeochemical consequence. Overall, this work will transform understanding of the underlying metabolic behavior of unicellular life at the base of the Universal Tree of Life and at the base of dark ecosystems. The objective of the proposed research is to determine whether microbial metabolic rate/yield trade-offs of hydrogenotrophic NO3--, Fe3+- and CO2-reducing microorganisms exhibit predictable adaptations to temperature and to the oxidation-reduction (redox) chemistry of catabolism during chemosynthesis. Specifically, we will test the hypotheses that (i) microbial metabolic rate-to-yield ratios will increase for all proposed microbial processes when at higher temperatures and that (ii) microbes relying on generally less exergonic catabolic redox reactions will systematically display higher anabolic efficiencies than those relying on more exergonic catabolic redox reactions. To test these hypotheses, the project will employ experimental microbiological and geochemical approaches to compare microbial respiration rates, cell (and total biomass) yields, and cell doubling times of different anaerobic chemosynthetic microorganisms during batch and continuous growth. The resulting metabolic and environmental data will demonstrate how microbial metabolic behaviors are shaped by their abiotic environments. 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.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary Hundreds of taste buds (TBs) are located in the epithelial trenches of the circumvallate taste papilla (CVP) at the posterior midline of the tongue. Each TB contains taste receptor cells (TRCs) that transduce taste information to the brain and are continually replaced from stem/progenitor cells outside of TBs. Cancer patients undergoing chemo- or radiotherapy often experience dysgeusia, or taste dysfunction, likely due to perturbation to TRC renewal. Patients experiencing dysgeusia have increased risk of depression, malnutrition, and poor treatment outcomes. Therefore, understanding the mechanisms regulating maintenance of taste epithelium and TRC renewal will inform development of treatments to prevent or restore taste loss in these patients. CVP homeostasis occurs through proliferation and differentiation of progenitor cells that generate both TRCs and the surrounding non-taste epithelium. Our lab has recently identified new, long-term SOX9+ progenitors that, as a population, generate TRCs and non-taste epithelium (NTE); however, the potential of individual SOX9+ progenitors is unexplored. SOX9+ progenitors are located distant from taste buds, in the epithelial junction linking CVP trenches to the ducts of the underlying von Ebner's minor salivary glands (VEG). Each CVP contains a dozen or more junctions containing SOX9+ cells, suggesting that multiple independent progenitor reservoirs contribute to taste epithelium. Additionally, in contrast to the highly proliferative CVP, the junction contains few proliferative cells, indicating SOX9+ cells are slow cycling. These findings lead to my hypothesis that individual SOX9+ progenitors from multiple junctions produce progeny that move into local CVP trench epithelium, become highly proliferative, and differentiate into TRCs and NTE. Thus, in Aim 1, I will use sparse SOX9 lineage trace, whole-tissue clearing, and EdU birth dating, to determine the lineage and proliferative potential of individual progenitors from multiple junctions. Our previous work has focused on SOX9+ progenitor contribution in adult homeostasis, but if and when these taste stem cells are established embryonically is unknown. Embryonic immunostaining reveals SOX9+ cells are present during initial formation of the CVP and have overlapping expression with Ptch, the Shh signaling receptor. Shh is necessary for anterior tongue taste bud development, and proper CVP trench invagination in the posterior tongue. These findings lead to my second hypothesis that embryonic Shh signaling is required to establish adult SOX9+ taste progenitors. In Aim 2, I will use embryonic lineage tracing and genetic deletion of Shh signaling in SOX9+ cells to determine when SOX9+ cells are established, begin contributing to TBs, and if Shh signaling is required in these processes. Overall, this work may lead to approaches to leverage concentrated pools of taste stem cells to restore taste function in patients.

NSF

This project explores how macrodomain proteins have evolved to balance their different multiple biochemical activities. Macrodomains are ancient enzymes that bind to and remove ADP-ribose, an important post-translation modification that is critical in several cellular stress responses, including DNA damage, ER stress, and virus infection. These enzymes are conserved through all domains of life, including archaea, bacteria, eukaryotes, and viruses, indicating that they are critical for multiple cellular processes. However, the function of macrodomain enzymes in cell biology and microbiology are just now being uncovered. Furthermore, recent research indicates that macrodomains have evolved to properly balance their biochemical functions depending on if they are expressed from a virus, a bacteria, or from a eukaryotic cell. This project will evaluate how viral macrodomains have evolved to develop the ideal biochemical properties that allow them function in the context of a virus infection. This project also has strong educational and community outreach components. Most notably, students at all training levels will participate in this project and will learn how to evaluate the evolution of proteins through workshops in phylogenetics. This project will provide new insights into the fundamental biology of macrodomain enzymes and could lead to new insights into antiviral drug-development. This project aims to define how the coronavirus macrodomain has evolved to best function in the context of a virus infection using the mouse coronavirus, murine hepatitis virus (MHV), as a model. The use of MHV, which is unable to infect humans, for creating macrodomain mutations eliminates the potential for gain-of-function research. Research over the last decade has demonstrated that the coronavirus macrodomain blocks innate immune responses, is critical for viral pathogenesis, and is a potential drug target. Furthermore, the macrodomain uses both its ADP-ribose binding and hydrolysis activities to promote virus replication, and they must be balanced for optimal replication. The PI will use recombinant proteins and a panel of recombinant viruses, developed using a bacterial artificial chromosome based reverse genetic system, to better understand how highly conserved amino acids and selective pressure induced mutations impact Mac1 biochemical activities, virus replication, and pathogenesis using well-established assays and model systems. Additionally, macrodomains from across the evolutionary spectrum will be expressed in the context of MHV infection to define the evolutionary limits of macrodomain divergence that is tolerated in MHV. This work will provide a deeper understanding of how macrodomains have evolved to counter various ADP-ribose dependent antiviral responses. This project is jointly funded by the Genetic Mechanisms Program and the Division of Molecular and Cellular Biosciences. 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.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Pneumonic plague is the deadliest form of disease caused by Yersinia pestis. Disease manifests as a rapidly progressing and highly lethal necrotic pneumonia that is typically fatal within seven days. If antibiotic treatment is not administered within 24 hours after the onset of symptoms, pneumonic plague is 100% fatal. The lethality of plague is due to the abrupt onset of a hyper-inflammatory response that compromises pulmonary function. Deletion of the Y. pestis inner membrane protein YbtX results in decreased neutrophil infiltration into the airways and decreased expression of select pro-inflammatory cytokines/chemokines during the later stages of pneumonic plague. YbtX is part of a zinc acquisition system by which the Y. pestis siderophore Yersiniabactin (Ybt) competes with host protein calprotectin to scavenges bioavailable zinc. YbtX acts as an inner membrane transporter that facilitates transport of Ybt-Zn into the bacterial cytosol. Calprotectin is also known to be a driver of host inflammatory responses. We hypothesize that YbtX-mediated zinc import contributes to the onset inflammation in the lung by activating calprotectin-dependent inflammatory responses. To test this, we will characterize how zinc depletion impacts inflammation during infection in the absence of YbtX. Further, we will alter the balance of host zinc/calprotectin in a murine intranasal infection model of pneumonic plague and characterize how this balance affects pulmonary inflammation. This work will determine how zinc and host calprotectin impact host inflammatory responses during pneumonic plague.

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This posting is to provide public notice of NPS' intention to fund the following project activities without full and open competition: Task Agreement P14AC01558 under Cooperative Agreement H8W07110001 with the University of Washington, a partner under the Pacific Northwest Cooperative Ecosystem Studies Unit, for the project titled, "Determining if Managed Wildfires and Prescribed Fires Conserve Critical Habitat Structure for Pacific Fishers in the Southern Sierra Nevada."

NIA - National Institute on Aging

Project Summary Abstract Alzheimer disease (AD) is the most common form of dementia that has no cure and few therapeutic options, and the strongest risk factor for developing AD dementia is aging, an unavoidable event. T cells are greatly affected during aging as they become comprised of less naïve/resting cells and more terminal differentiated/senescent cells that can be cytotoxic and produce high amounts of pro-inflammatory cytokines. Inflammation is known to be an early event in AD with innate microglial activation and an augmented cytokine production in the CNS and increases in frequency of circulating Th1, TH17 and Th9 inflammatory T-cells. Functioning in distinct contrast to these inflammatory T cells, are regulatory T cells (Tregs) which are a small, usually stable, lineage of inhibitory CD4 T cells that expresses the FoxP3 transcription factor and is essential for maintaining healthy homeostasis and tolerance. Tregs appear to be involved in AD pathology as most reports have found them to be at low frequency in the AD patient circulation, while mouse AD models demonstrate that Tregs can affect the disease as the adoptive transfer of Tregs slows disease progression. The ability of Tregs to down modulate inflammation makes them central to immune resolution and inhibition of autoimmunity. But Tregs have additional far-reaching activities as they not only can inhibit the activation of microglia and other immune lineages (B, T, monocyte), but also can initiate tissue repair and recovery. Tregs have been shown to be neuroprotective in mouse models for brain hemorrhage and for the neurodegenerative diseases Parkinson’s, ALS, and AD. Excitingly, the administration of wild type Tregs into the APPPS1 AD mouse model was found to slow disease and also induce recovery of cognition. Yet, only one group has actually isolated and performed the in vitro functional assays needed to determine if AD patient-derived Tregs are functionally competent or if their reduced activity would represent a potential therapeutic target. Unfortunately, this study used suboptimal Treg isolation methods, combined functionally-distinct Treg subsets into a single population, and analyzed only one parameter to assess Treg function – suppression of target cell proliferation. Thus, it is crucial that a more comprehensive study be performed to reveal the breadth of potential dysfunction of Tregs in AD-dementia and expose dysfunction-inducing mechanisms. As our lab routinely examines human Treg function in disease, we have begun to study AD-Tregs using a high stringency isolation and multi-parameter functional assay approach that separately interrogates functionally distinct subsets of Tregs isolated from pairs of matched AD-dementia vs HC PBMCs. Our overarching goal is to uncover if and why Tregs show dysfunction in AD. Illuminating the mechanisms of Treg deficiency in AD will contribute to future development of Treg-directed therapies for AD, where optimal Tregs may slow progression, induce tissue repair and restore cognition.

NCI - National Cancer Institute

PROJECT SUMMARY Colorectal Cancer (CRC) is a significant burden on human health and in need of better diagnostics and treatments. Microbes have been implicated in ~20% of all human cancers, and recent studies have shown strong associations of CRC with bacteria residing in the gut. The gut microbiota has well-established roles in modulating intestinal immune responses that can exacerbate or alleviate inflammation, however, the ways in which members of the gut microbiota modulate pro or anti-tumor immune responses remain largely unknown. Colonic tumors are infiltrated by surrounding immune cells and disease outcome is heavily influenced by which immune cells are recruited to tumors and their roles within the tumor microenvironment (TME), therefore, understanding the mechanisms of how gut bacteria can modulate those infiltrating immune cells is critical in designing new and more improved diagnostics and interventions. In my work investigating human microbiotas from CRC patients, I discovered a human gut commensal, Bacteroides uniformis, protects against disease by enhancing anti-tumor immunity. This protection is dependent on Natural Killer (NK) cells and is effective in multiple pre-clinical mouse tumor models. I found that B. uniformis directly activates NK cells, and this effect is independent of adaptive immunity. However, the bacterial factors recognized by NKs are still unknown, as are the dynamics within the NK cells after B. uniformis stimulation in the gut and TME. B. uniformis and other Bacteroides spp. can modulate immune responses via their unique capsule polysaccharides, but their impact on anti-tumor immunity is unknown. Therefore, I hypothesize that B. uniformis directly activates NK cells, through its capsule molecules, resulting in remodeling of the immune landscape in the tumor microenvironment, and enhancing anti- tumor immunity to protect against CRC. I will investigate this hypothesis along three lines of interrogation. Specific Aim 1: Identify the bacterial factor by which B. uniformis modulates NK cell activation and cytotoxicity. Specific Aim 2: Determine how strain level genetic variations in B. uniformis influences immune activation and CRC development. Specific Aim 3: Investigate how bacterial induced NK activation in the tumor microenvironment promotes anti-tumor immunity. By completion of this proposed work I will have a more nuanced understanding of the mechanisms by which B. uniformis prevents tumor formation via NK cells in the TME, which will lead to translational progress in innovative diagnostics and novel therapies for patients with CRC.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Exercise is an important modulator of human health, and mechanical loading during strength and resistance- based exercise is well known to positively impact bone mass by enhancing osteoblast-mediated bone formation. However, there is a fundamental gap in understanding the response of bone to aerobic metabolic demands found in activities like running and cycling, and how those demands interact with the mechanical loads placed on the skeleton during exercise to differentially modulate bone mass. Emerging evidence shows that aerobic exercise induces acute elevations in the bone resorption marker carboxy-terminal telopeptide of type I collagen (CTX) without compensatory changes in bone formation, suggesting an immediate and unexplained pro-resorptive response. While well-documented, there is substantial lack of prior rigor regarding the cell source, extent, and purpose of elevated bone resorption during aerobic exercise. These questions are important because they may help facilitate the development of more precise recommendations for aerobic exercise to modulate bone health. Furthermore, they may provide greater understanding of factors governing bone loss in conditions of high metabolic demands or low energy availability that often result in fractures or additional disability in vulnerable populations. The central hypotheses of this proposal are that the source of CTX elevation during exercise exists on a spectrum between osteocyte perilacunar remodeling (PLR) and osteoclast resorption, the extent of which is governed by energy demands during exercise, and that CTX elevation is proportional to amino acid flux into skeletal muscle. These hypotheses will be tested in three distinct, non-overlapping specific aims. Aim 1 will determine the source of elevated CTX during aerobic exercise. We will selectively inhibit osteocyte perilacunar remodeling using MMP13ocy-/- mice and compare exercise-induced elevations in CTX to mice treated with pamidronate, which we have previously shown can inhibit osteoclast activity in the face of sustained PLR, or cathepsin K inhibitor, which selectively inhibits collagen degradation, but not demineralization of the bone ECM. Aim 2 will determine the relationship between CTX elevation and energy demands during exercise. We will run well-trained human subjects, and rats bred for high and low aerobic capacity, at treadmill speeds correlating with varying oxidative and non-oxidative energy demands. Subjects will run on commercial (human) and custom (rat) treadmill-interface systems to uncouple metabolic demands from biomechanical loading, and we will further modulate energy demands in the presence or absence of carbohydrate supplementation. Lastly, Aim 3 will evaluate the relationship between CTX elevation and amino acid distribution to skeletal muscle during aerobic exercise. We will assay circulating and muscle-targeted amino acids in rodent samples to quantify amino acid flux to the muscle in relation to both modulation of bone turnover and energy demands of the exercise itself.

NCI - National Cancer Institute

Glioblastoma (GBM) is an aggressive brain tumor that recurs in treatment-refractory form within months of initial standard-of-care (SOC) treatment. Extensive study of primary (pre-treatment) GBM has revealed numerous characteristics that impede treatment, including marked intratumoral genetic, transcriptional, and spatial heterogeneity; rapid infiltration throughout the brain parenchyma; a uniquely compromised immune microenvironment; and immunosuppression beyond the central nervous system. However, the mechanisms by which these factors enable disease progression are not understood, and the critical disease entity -- recurrent GBM -- is comparatively understudied. The central hypothesis of this study is that identifying the unique characteristics of recurrent GBM and the mechanisms driving its evolution from primary GBM will reveal new therapeutic targets. Our overall objective is to identify transcriptional, genetic, neuronal, and immunological factors that influence GBM formation, progression, and treatment response, with an emphasis on therapeutically targetable ligand-receptor interactions. We utilize unique human tissue repositories, novel experimental designs, and spatially-resolved single-cell approaches with the rationale that reversible interactions between tumor cells and diverse nonmalignant cells drive tumor formation, progression, and drug resistance. We pursue our objective through three Specific Aims: (1) Identify unique molecular, cellular, and spatial features of recurrent human GBM; (2) Identify mechanisms underlying distal pathological effects of GBM, including distant brain infiltration and systemic immunosuppression, in whole-brain and whole-body autopsy settings; and (3) Elucidate the dynamic reconfiguration of the GBM ecosystem during tumor formation and treatment response using mouse models, single-cell lineage tracing, and spatial transcriptomics. In Aim 1, we will integrate spatial transcriptomics (ST) with novel long-read single-nucleus RNA-sequencing (snRNA-seq) and exome sequencing to identify cell states, single-cell clonal architecture, multicellular niches, and heterotypic ligand-receptor interactions specific to recurrent GBM. In Aim 2, we will leverage unique resources at MGH that enable the analysis of diverse brain regions and peripheral immune organs in post-mortem GBM patients. We will combine snRNA-seq, ST, and single-cell T-cell receptor sequencing to study brain invasion and immunosuppression within the brain and peripheral immune system. In Aim 3, we will perform time-resolved single-cell RNA-seq (scRNA-seq) and ST in a mouse model of GBM to map the temporal and spatial co-evolution of tumor and immune cells during tumor development and response to SOC. Critically, we will integrate single-cell lineage tracing with scRNA-seq and ST to distinguish among possible mechanisms by which treatment reconfigures the tumor ecosystem. This study addresses innovative questions using unique resources, novel study designs, and new combinations of genomic and computational approaches. The work is significant because it will enable the discovery of therapeutic vulnerabilities associated with GBM development, progression, and recurrence.

NIH

Custom accommodative insoles are prescribed to patients with diabetes who are at risk of developing plantar ulcers. Custom insoles aim to reduce plantar pressures through an accommodating insole with a weight-bearing surface that conforms to the patient’s foot. The current standard of care (SoC) insoles are typically constructed from an EVA foam block milled to match a patient’s foot shape, captured via a foam crush box, and then covered with a uniform sheet of poron and plastazote foam. The material properties of these insoles are generic and not patient specific. We have developed insoles that not only have patient specific shape but also patient specific regions of modulated stiffness. Our insoles are more effective in reducing plantar pressure in the areas of high pressure than the current SoC insoles in a healthy population and investigation is underway with similar preliminary results in individuals at risk of developing a plantar ulcer. Our work suggests that both foot shape and plantar pressure are important factors when designing accommodative insoles and offer the clinician a new tool to better customize the insole to the patient’s specific needs. Clinically, direct foot-shape scanners are already being implemented. However, it is critical to understand if these scanners distort the shape of the foot compared to the traditional foam crush box and how reliable the various scanning methods are within and across clinicians. Evaluations of intra-rater reliability of basic measures of foot shape (i.e., length and width) have been conducted, however, more comprehensive methods evaluating the plantar surface such as arch volume and overall surface differences are critical when designing an insole, given that the insole must conform to the foot shape. Thus, the purpose of this proposal is to evaluate the methods for obtaining shape and their effects on accommodative insole design and the ability to offload high-pressure areas in people with diabetes. Additionally, plantar pressure is not incorporated into the clinical design of SoC insoles. Compared to our current in-shoe pressure capture method, barefoot walking plantar pressure is a much more practical metric for clinicians to obtain. In this project, we will also determine if designing insoles with barefoot or in-shoe plantar pressure yields similar reductions in plantar pressure. Aim 1a: Quantify the inter- and intra-clinician reliability of three methods of capturing foot shape (crush box, Tiger scanner, structure sensor). Aim 1b: Quantify the differences in foot shape obtained using three methods of capturing foot shape. Aim 2: Evaluate the ability of insoles designed using the various foot scanning methods to offload areas of high pressure in the diabetic foot. Methods: Aims 1a and b: Three clinicians will take three shape measurements of the feet of ten healthy individuals with each of the following foot shape capture devices. Standard 1-D measurements and more comprehensive methods of evaluating the plantar surface will be calculated. Inter- and intra-clinician reliability will be calculated for each of the foot-shape capture methods (Aim 1a) and differences in the overall foot shape across devices compared (Aim 1b). Aim 2: 20 individuals with diabetes and elevated plantar pressures will undergo a clinical foot exam, have foot shape captured (using the three techniques described above), and have plantar pressures recorded during barefoot and shod walking. Seven sets of insoles will be manufactured for each participant, one pair of the SoC insole and six pairs of 3D printed insoles using the various foot shape and pressure capture methods. In-shoe plantar pressure will be measured during walking while wearing the insoles. Participants will also complete a comfort and usability survey after each insole condition. Peak pressure and pressure time integral in four regions (first metatarsal head, second metatarsal head, third to fifth metatarsal head, and offloading region) of the foot will be compared. VHA currently cares for 563,000 Veterans defined by the Prevention of Amputation in Veterans Everywhere (PAVE) Program as being at high risk for ulceration and amputation. Developing custom offloading insoles that are more efficacious than the current standard of care by leveraging advances in 3D printing has the potential to reduce rates of amputation and morbidity in our Veteran population.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Protein targeting is essential for maintaining cellular organization and function across the tree of life. Signal Recognition Particle (SRP) is a ribonucleoprotein complex that plays an important role in this process, co- translationally targeting 30% of the proteome for membrane insertion or secretion. SRP recognizes the signal sequence of secretory proteins, pauses translation, and guides the ribosome-nascent chain complex to the endoplasmic reticulum. Errors in SRP-dependent protein targeting lead to protein aggregation and fitness defects, and are associated with developmental disorders, neurodegeneration, and cancer. Despite SRP’s important function, the mechanisms underlying its assembly remain poorly understood. Specifically, the factors involved in SRP assembly and how assembly is subcellularly coordinated remains largely unknown. Therefore, the goal of this proposal is to identify SRP assembly factors and characterize their mechanism in human cells. Structurally, SRP consists of six proteins and a 300-nucleotide noncoding RNA, 7SL. Using microscopy, we and others have observed that several of these components localize to nucleoli, which are biomolecular condensates that form through liquid-liquid phase separation and facilitate ribosome biogenesis. Based on these observations, I hypothesize that nucleoli concentrate factors that mediate 7SL maturation, which is necessary for SRP assembly. This will be tested through two specific aims. In Aim 1, I will identify 7SL RNA-binding proteins and investigate their role in SRP assembly and 7SL folding. In Aim 2, I will employ CRISPR screening to identify nucleolar SRP assembly factors and determine whether their condensation contributes to function. By testing whether nucleoli coordinate SRP assembly, this work will advance our understanding of biomolecular condensates and ribonucleoprotein biogenesis, revealing novel therapeutic targets for diseases that arise from protein mistargeting. Furthermore, this study constitutes the research arm of a comprehensive training plan that facilitates my development as a physician-scientist. Throughout this proposal, I will have mentored training in molecular biology research and gain the skills necessary to become an independent investigator, including experimental design, scientific writing, and presentation skills. This work will be conducted at the Omenn-Darling Bioengineering Institute at Princeton University, which has state-of-the-art microscopy, flow cytometry, and genomics equipment. My research training will be complemented with longitudinal clinical training in precision medicine and hematology-oncology at the Rutgers Cancer Institute. Together, my research and clinical training will provide a strong foundation for pursuing a career as an independent physician-scientist.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Coccidioidomycosis infections cause almost a third of community-acquired pneumonia cases in Arizona, and reported cases have doubled from 2013-2021. Coccidioidomycosis is an infection caused by inhalation of fungal spores from the soil-dwelling Coccidioides genus, and can lead to chronic lung infection, meningitis, or death. The disease is a growing public health concern in the southwestern US, particularly in Arizona, which reports around 10,000 cases per year—nearly two-thirds of US cases. The disease imposes a significant public health and economic burden, with Arizona’s 2019 cases alone estimated to cost $736 million in lifetime expenses. Yet, public health responses have been hindered by critical gaps in understanding of the environmental drivers of infection, particularly the role of dust exposure. Changing hydrometeorological factors, such as increasing aridity and more frequent dust storms, may be contributing to rising incidence, as dust and dust-generating processes (e.g., wind erosion, construction, agriculture) likely promote aerosolization and dispersion of Coccidioides spores. However, the relationship between dust exposure and coccidioidomycosis incidence remains unclear, with some studies linking short-term, extreme dust events to outbreaks and others finding no impact. This project aims to fill these knowledge gaps by leveraging a novel database of spatiotemporally refined dust concentration estimates and all reported coccidioidomycosis cases (n > 126,000) in Arizona from 2005 to 2022. Our secondary data analysis study has three aims: 1) Estimate the effects of daily ambient dust exposure on coccidioidomycosis incidence and identify populations most vulnerable to these exposures, focusing on determinants of social vulnerability such as low income, poor housing quality, and racial/ethnic minorities; 2) Assess the impact of extreme dust events on coccidioidomycosis incidence, and identify populations most vulnerable to these exposures; 3) Investigate whether hydrometeorological factors, including oscillations between wet and dry conditions that may influence spore growth and dispersal, modify the relationship between dust exposures and coccidioidomycosis incidence. Our results will help guide public health messaging and target prevention strategies in Arizona to both the general population and clinicians surrounding when and where risk of coccidioidomycosis transmission is highest; provide important evidence on the contribution of environmental change to transmission; and enable identification of populations and geographies particularly sensitive to dust exposures.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary Metabolic Syndrome (MetS) affects over one-third of U.S. adults and is closely linked with oral health issues, particularly periodontitis. The bidirectional relationship between MetS and periodontitis exacerbates both conditions, but the underlying mechanisms remain unclear. Our preliminary R03 data indicate that MetS alters the oral microbiome, creating an ‘at-risk-for-harm’ environment even in the absence of clinical periodontal disease. This project aims to elucidate these mechanisms through a longitudinal systems biology approach, focusing on how restoring metabolic health impacts the functional oral ecosystem. We hypothesize that improved metabolic health will shift the oral microbiome towards a eubiotic state, reduce pathogenic strains, and alter the metabolome to a less inflammatory profile. We test our hypothesis in the following specific aims. In Specific Aim 1: We will track the oral microbiome's temporal dynamics in MetS and Metabolically Healthy Obese (MHO) cohorts undergoing lifestyle or surgical interventions, comparing them with healthy and periodontal disease cohorts. In Specific Aim 2: We will analyze changes in the oral metabolome post-metabolic intervention using advanced mass spectrometry techniques. In Specific Aim 3: We aim to identify and validate oral biomarkers and risk indicators linked to metabolic health improvements. This study will use a causal mediation framework to explore direct and indirect effects, providing crucial insights into the MetS-periodontal health relationship and aiding in the development of prognostic biomarkers and risk indicators in the future.

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This funding opportunity is to provide public notice of NPS's intention to fund the following project activities without full and open competition. Task Agreement # P15AC00255 under Cooperative Agreement P11AC8R001 with the University of Nevada, Reno, a partner under the Californian Cooperative Ecosystem Studies Unit, for project titled "Determining the Influence of a Predaceous Diving Beetle (Neoclypeodytes cinctellus) on the Devils Hole pupfish (Cyprinodon diabolis) and its Prey"

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Whether due to injury, disease, or as a consequence of cancer chemotherapy, millions of Americans, both young and old, experience sensory neuropathy. This condition, characterized by numbness, tingling, and disruptions to gait, degrades the quality of life. The sheer diversity of cells that underpin the mechanical senses of touch, hearing, and proprioception pose a barrier to understanding their function in health and their dysfunction in disease. Rapid mechanosensation responsible for low-threshold touch sensation depends on the activation of specialized mechanosensitive (MS) ion channels. The proteins forming MS channels in humans and other animals have come to light over the past two decades, yet how mechanical energy causes their activation remains poorly understood. Some MS channels seem to respond to changes in membrane tension, others depend on coupling to the cytoskeleton, and still others require coupling to extracellular matrix (ECM) structures. Whereas a subset of neuropathies arise from faulty nerve transmission, many other forms involve defects in coupling MS channels to sensory stimulation or from defects in the channels themselves. This project seeks to understand how sensory stimuli is coupled to MS channel activation in vivo. Very few biological models are amenable to in vivo neuronal biophysics, including voltage-clamp of sensory neurons and to imaging MS channel complexes in living animals. The model organism C. elegans fulfills these requirements. In this research we will combine established techniques for in vivo whole-cell patch-clamp electrophysiology with cutting-edge, genetically encoded tools to visualize and measure touch-induced mechanical strain in touch receptor neurons. A large toolbox of existing mutations affecting the cytoskeleton and ECM will be used to perturb the coupling between sensory stimulation and MS channel activation, delineating the role played by each structure in this fundamental sensory process. Ample evidence indicates that the mechanics of touch are conserved even among distantly related animals, suggesting that what is learned through this research training plan has the potential to provide insight into the factors that give rise to sensory neuropathy and to other mechanosensory abnormalities.