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NIGMS - National Institute of General Medical Sciences

Integrated mass spectrometry strategies to decipher protein-metabolite cross regulation Cellular metabolism relies on protein enzymes to convert substrates to products, working in interconnected networks to generate important biological precursors, remove toxic waste, balance redox and defend energy potential. However, the study of metabolic regulation in both healthy and disease states has proven challenging in part due to the intricate cross-regulation between metabolites and proteins. Current strategies to characterize metabolic regulation have generally focused on characterizing metabolites or protein enzymes, but not both. Consequently, how metabolites and proteins interact in the cellular context remains an open question in the field of metabolism and cellular biology. To bridge this gap, the Skinner lab is focused on characterizing the interactions between metabolites and proteins in the cellular context by using mass spectrometry to integrate existing multi-tiered methods in addition to developing new techniques to enhance the characterization and scope of both protein and metabolite measurements. Our primary research focus is on the regulation of proteins and metabolism by redox cofactors through signaling of sulfur-containing metabolites (Area 1) and the effect of vitamin B6 on metabolic and redox regulation as it is converted to protein cofactor pyridoxal-5-phosphate (PLP) (Area 2). These two areas each represent critical intersections of metabolome and proteome that play a key role across a variety of disease states. To better understand these two ranges of metabolite and protein cross-regulation, we will apply top-down proteomics and native mass spectrometry analyses of intact protein complexes coupled with full-scan and stable isotope labeling metabolomics to simultaneously probe metabolites and proteins. In Area 1, we will characterize how the abundance and redox state of specific thiol metabolites and cellular nicotinamide adenine dinucleotide phosphate (NADP+) redox state can modulate glycolysis and other core metabolic pathways Protein thiols have been demonstrated to be covalently modified at high stoichiometry with a variety of different groups, making this a likely axis for metabolic regulation. In Area 2, we will examine another key intersection between metabolite and protein regulation; the metabolism of vitamin B6 to its active form PLP prior to acting as a cofactor for 53 human enzymes. Specifically, we will characterize how some PLP-containing enzymes seem more resistant to extended B6 deficiency than others and how changes in subcellular nicotinamide adenine dinucleotide (NAD+) redox state can affect PLP levels and vice versa. This project integrates metabolomics with native mass spectrometry and proteomics and applies it to two pressing questions in the field of systems biology and regulation that lie at the intersection of metabolome and proteome.

NIGMS - National Institute of General Medical Sciences

Abstract Functional development of the body plan and organs requires tissues to adopt the specific three- dimensional morphologies required for function. Defects in tissue shape lead to functional deficits with impacts ranging from reduced or absent organ function to peri-natal lethality. The complex tissue shapes required for function are built from simple precursors by the spatially and temporally coordinated activity of individual cells. In many cases, final tissue shape is driven exclusively, or nearly exclusively by a single class of cell dynamic, such as cell shape remodeling, rearrangement of cells with respect to one another, or localized or oriented cell divisions. However, many tissues rely on a combination of multiple behaviors to build complex morphologies, which presents a challenge as these behaviors are incompatible within individual cells. Thus, a major challenge in human health and developmental biology is to understand how multiple classes of cell dynamics occurring concurrently in the tissue are successfully integrated despite their mutual antagonism in individual cells. A key related question is how these disparate behaviors are controlled and spatially patterned within tissues, as this spatial control is key to successful integration. In this proposal, we will use the mouse cranial neural plate as a model system for understanding how behaviors are integrated. This is an attractive system, as at least three mutually disruptive behaviors occur: apical constriction, planar polarized cell intercalation, and oriented cell divisions. We will use a combination of mouse genetics, bespoke cellular and subcellular live imaging approaches, quantitative in toto imaging of tissue organization, and a combination of molecular and transcriptomic approaches. Together, these experiments will directly define the role of each class of cell behavior in generating final tissue shape, elucidate the spatially delimited developmental signaling systems that control individual cell behaviors, and determine how mutually disruptive behaviors are integrated to drive complex tissue shape outcomes.

Intel Foundation

Funds STEM education programs for K-12 students, with emphasis on increasing access for underrepresented groups in technology.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The increasing frequency of respiratory infectious diseases, such as influenza and COVID-19, which can spread rapidly and lead to severe outbreaks, necessitates that we re-envision our approaches to monitor pathogen exposures in the indoor environments. Current surveillance methods mostly depend on syndromic data from hospital admissions, clinic visits, and school absenteeism rates. However, these approaches can lead to underestimation and delay in disease surveillance due to no reporting of mild or asymptomatic cases, lack of access to healthcare, and time-consuming lab and diagnostic processes. A proactive approach in combating airborne diseases requires early detection of target pathogens. Here, we propose to innovate an intelligent system capable of real-time, efficient, and cost-effective monitoring of airborne pathogens in the environment. We will build upon our preliminary success in automated bioaerosol sampling and pathogen detection, to create an Airborne Pathogen Sensing (APS) system. This goal will be achieved by focusing on two specific aims. First, we will create a novel class of modular whole-cell biosensors for sensitive, rapid, and robust detection of multiple critical airborne pathogens. The pathogen detection will be achieved by creating quenchbody (Q-body) display biosensors, where target specific quenchbody is expressed and displayed on the surface of microbial host cells. When the Q-body binds to its antigen (target pathogen), the fluorescence intensity substantially increases via the antigen-dependent removal of the quenching effect on the fluorophore. Second, we will design and build a portable and automated bioaerosol sampling device that can be coupled to the biosensing and signal detection systems. To this end, we will evaluate and optimize two sampling devices: mist chamber and biosampler, and choose the device with the highest bioaerosol capture efficiency. Finally, we will integrate the biosensing component with the bioaerosol sampling device and an automated flow-through fluorescence detection system to achieve automated real-time monitoring of airborne pathogens.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract This research project delves into the complex biological phenomenon of organelle-specific Unfolded Protein Responses (UPRs) in eukaryotic cells, focusing on their role in maintaining proteostasis under stress conditions prevalent in aging. We aim to understand the intricate communication and coordination mechanisms between UPRs in various cellular compartments, such as mitochondria, the endoplasmic reticulum, and the cytosol. A key aspect of our study is the interplay between lipid metabolism and these protein-folding processes, an underexplored area in cellular biology. Lipid dyshomeostasis is a major contributor to a range of devastating diseases, including neurodegenerative diseases, cardiovascular disease, diabetes, and cancer. Despite extensive research, the role of lipids in regulating cellular homeostasis remains unclear. Our long-term goal is to understand how cells utilize different organelle stress responses to maintain homeostasis under various stressors. The overall objective of this project is to explore the connection between lipid homeostasis and proteostasis and their interplay in the context of organelle-to-organelle communication. Our central hypothesis is that the relative cardiolipin-to-ceramide ratio is crucial for communication between stress response pathways in the mitochondria, ER, and cytosol. This hypothesis is based on preliminary data generated from well- characterized C. elegans and mammalian cell culture systems. Our previous research found that a branch of unfolded protein response in mitochondria (UPRmt), triggered by mitochondrial protein folding stress, activated the cytosolic unfolded protein response (UPRcyt) while inhibiting the unfolded protein response in the ER (UPRER) to increase cellular resistance to proteotoxic stress. We also discovered a novel mechanism of stress response regulation involving the opposing effects of two lipids, cardiolipin and ceramides, on proteostasis. Using both C. elegans and mammalian cell culture systems, this project will employ cutting-edge techniques in proteomics, lipidomics, and advanced imaging to provide an efficacious and comprehensive view of these cellular processes. For the next five years, the research program aims to map pathways and interactions in organelle-specific UPRs and determine how these processes are spatiotemporally regulated under proteostasis stress, offering novel insights into the cellular maintenance of protein homeostasis and its impact on cellular and organismal health.

NIA - National Institute on Aging

Project Summary/Abstract Sepsis, the body’s injurious response to infection, afflicts nearly 50 million people worldwide annually with a particularly high burden in older patients. The majority of sepsis survivors experience prolonged or permanent brain dysfunction. We have recently discovered that this form of post-septic impairment is partially driven by a pathologic organ crosstalk between the systemic vasculature and brain, a process that may be exacerbated by age. The endothelial glycocalyx, a heparan sulfate (HS)-rich layer that lines the lumen of all blood vessels, is degraded during sepsis, releasing highly biologically active HS fragments into circulation. These circulating HS fragments selectively deposit within the hippocampus and can worsen cognitive outcomes in sepsis survivors. Strikingly, circulating HS levels are 5-fold higher in older (≥65) compared to younger patients (<50) with sepsis. This aging-associated increase in HS shedding may be due to a more fragile endothelial glycocalyx. In preliminary preclinical studies, we have found that increased age was associated with ultrastructural changes of the glycocalyx, including a loss of 6-O sulfation residues within endothelial-derived HS. Loss of 6-O sulfation is known to increase HS susceptibility to degradation by heparanase, the “sheddase” responsible for glycocalyx degradation during sepsis. This loss of sulfation may be due to increased circulating Sulfatase-2 (Sulf2) an enzyme dedicated to removal of 6-O sulfates from HS as 1) preliminary single cell RNA sequencing of peripheral blood mononuclear cells demonstrated that aged humans have increased levels of Sulf2 in monocytes and 2) aged mice exhibited increased blood Sulf2 activity. Based on these preliminary observations, we hypothesize that age-related remodeling of endothelial HS by monocyte-derived Sulf2 predisposes older patients to release brain-penetrating HS during sepsis, which will be associated with post-septic brain dysfunction. We will rigorously test this hypothesis in both pre-clinical models of sepsis and a prospective, observational cohort of patients with sepsis. We will specifically 1) determine whether age-related increases in expression of Sulf2 in monocytes are responsible for endothelial glycocalyx fragility in older mice; 2) determine whether age-related increases in expression of Sulf2 in monocytes exacerbate pathogenic HS deposition during sepsis in aged mice; and 3) identify whether circulating pathogenic HS fragments are associated with impairment in multiple cognitive domains in older sepsis survivors. This work will mechanistically investigate a unique form of age-related pathologic interorgan communication: release of brain-penetrating, pathogenic HS from the systemic vasculature. Critically, it may also identify Sulf2 inhibition as a therapeutic target in sepsis, which could improve the lives of millions of older survivors per year.

Areion Unmanned Aerial Systems LLC

Interpath flight tests

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY In the healthy intestine, the microbiota and intestinal immune system are intimately linked. One of the major regulators of the microbiome is the mucosal antibody, IgA, produced by intestinal B cells, which exerts unique immunomodulatory functions on bacterial communities. Failures in bacterial regulation by IgA has serious health implications, impacting the progression of a wide range of diseases like inflammatory bowel disease (IBD), obesity, cancer, and depression. However, whilst many studies have identified roles for IgA in bacterial regulation, how the presence of the microbiota directs differential programming of local B cell responses towards production of regulatory IgA is poorly understood. Most homeostatic IgA is produced via gut-associated germinal centers (gGCs), microanatomical niches which are essential for B cell and antibody evolution. Although we have previously shown that gGCs support effective selection of microbiota-specific B cell clones, accumulating evidence indicates that fundamentally altered signaling networks govern the dynamics of steady-state gGCs. In our preliminary data we identify composition of the microbiota as a significant modulator of several aspects of gGC responses, suggesting a crosstalk that is established between the intestine, B cells, and the microbiota. We therefore hypothesize that ‘education’ in the intestinal microenvironment by the microbiome fundamentally alters how B cells to evolve in gGCs. We show evidence that the responsiveness of several immunoreceptors is modified following exposure to the microbiome, suggesting that chronic bacterial stimulation broadly desensitizes receptor function and sets lowered activation thresholds. Commensals are perceived by cells specifically through the B cell receptor (BCR) and via generic sensing through Toll-like receptors (TLRs). We present new evidence supporting that sustained stimulation by TLR ligands acts as a signal rheostat, tuning inherent responsiveness of B cell receptor and CD40 signaling networks to regulate permissive entry to chronic gGCs. Consequentially, TLR deficient animals host enlarged gGCs, in direct opposition to inflammation-driven GCs, which are severely compromised in the absence of TLR activation. We propose a model whereby continuous low-level TLR signaling in combination with weak BCR binding could be sufficient to initiate gGCs towards the microbiome, facilitating the selection of a regulatory pool of IgA with broad binding specificity. We will test our hypotheses through use of two specialized imaging approaches, a novel commensal-specific transgenic BCR mouse model, and restricted-diversity microbiomes, to address how exposure to microbial signals tunes the magnitude and specificity of intestinal B cell responses. Our studies will further our understanding of the mechanisms driving dysregulation of the microbiome, important for autoimmunity, cancer, and metabolic disorders. Results from our proposal will also generate valuable insights into the rules governing tissue-specific antibody responses and IgA memory, a rapidly emerging interest area in mucosal infectious disease research.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT: Our laboratory discovered and confirmed STING (Stimulator of Interferon Genes) as an essential component of the cytosolic DNA-mediated innate immune response pathway. We subsequently demonstrated that STING signaling is commonly suppressed in microbial infected cells or human cancers, which enable such cells to escape the host immunosurveillance system. Our new data, enclosed herein, indicates that intrinsic innate immune STING signaling stimulates the production of essential innate immune proteins that in combination with microbial or self-nucleic acid, renders such cells highly immunogenic. Here, we intend to clarify the mechanisms of how intrinsic STING signaling facilitates the trans-activation of phagocytes and the cross-presentation of microbial or tumor antigen. Aim I: We aim to evaluate the mechanisms by which innate immune STING signaling renders microbial infected cells or DNA-damaged cells immunogenic, compared to normal apoptotic cells (which are non-inflammatory). Our data indicates that cytosolic DNA triggered STING-inducible genes combine with and protect, cytosolic DNA species to enable them to escape DNase-mediated degradation and activate STING signaling, in trans, in engulfing phagocytes. We have now identified these genes and will further characterize their novel mechanisms of action. Aim II. Our data indicates that innate immune STING signaling is suppressed in aged cells by epigenetic silencing. Such cells, for example, STING -/- MEFs or transformed cells lacking cGAS or STING expression, are non- immunogenic following DNA-damaging events, even though they produce cytoplasmic micronuclei. We hypothesize that reconstituting intrinsic STING signaling in vitro and, in vivo, will render cells immunogenic and trigger the innate activation of engulfing phagocytes. We believe that these strategies could shed insight into mechanisms of resistance to cancer therapies, (many of which invoke micronuclei formation) as well as lead to new strategies to help improve the treatment of a wide array of inflammatory and malignant disease.

NIA - National Institute on Aging

Frontotemporal dementia caused by mutations in microtubule-associated protein tau (MAPT), including the N279K mutation, is a common cause of early-onset dementia. It is neuropathologically characterized by toxic aggregation of hyperphosphorylated tau, glial activation, and neurodegeneration. The factors contributing to the disease are likely numerous and poorly understood, and no disease-modifying therapies exist for FTD. Oxidative stress (OS) occurs when a cell’s innate antioxidant system is overwhelmed by reactive oxygen species, and oxidative modifications of biological molecules have important consequences on protein, DNA, and lipid function. In particular, uncontrolled lipid peroxidation can lead to ferroptosis, a specific cell death pathway which we found to be enriched in FTD postmortem brain and may contribute to neurodegeneration. We also identified an OS and neuroinflammatory phenotype in postmortem brain from FTD patients and induced pluripotent stem cell (iPSC)-derived neurons from FTD patients. Specifically, FTD iPSC-derived neurons show upregulation of the gene secreted phoshoprotein-1 (SPP1) and its protein product osteopontin (OPN), which can activate iPSC-derived microglia in vitro. Given the centrality of OS in our FTD models and the apparent association with SPP1, this proposal seeks to investigate mechanisms of OS generation and downstream sequelae in FTD. In aim 1, I will interrogate the effects of different classes of oxidative and ferroptotic stressors on FTD MAPT N279K iPSC-derived neurons. In aim 1a I will assess cell viability and lipid peroxidation. In aim 1b I will assess tau pathology and neurite outgrowth. In aim 1c I will attempt to rescue any effects seen in aims 1a and 1b by co-treating with antioxidant and ferroptosis inhibiting compounds. In aim 2 I will characterize astrocyte-neuron crosstalk in the FTD context. First, in aim 2a I will generate iPSC-derived astrocytes from FTD MAPT N279K patients or healthy control patients and treat with OPN and assess for astrocyte reactivity. In aim 2b I will generate antioxidant response gene reporter astrocytes and treat with Ctrl or FTD neuron conditioned medium to determine the role of neuron-secreted factors in astrocyte response. Finally, in aim 3 I will explore the potential of targeting OS in FTD. I will xenotransplant FTD or Ctrl neural progenitor cells into mice forebrains and treat systemically with liproxstatin, an antioxidant and ferroptosis inhibiting compound. In aim 3a I will characterize proteins involved in these pathways as well as glial reactivity and graft survival by histology. In aim 3b I will perform snRNA-seq on micro dissected grafts to map changes in gene expression profiles in response to OS targeting.

NIGMS - National Institute of General Medical Sciences

Project Summary / Abstract Animal behavior manifests from a coalition of physiological states to enable effective navigation of a world which perpetually challenges homeostatic regulation. Whereas homeostatic feedback regimes maintain physiological thresholds, allostasis describes the flexible adjustment of thresholds during the experience of novel stressors. To achieve whole-body allostasis, multi-organ physiology must be coordinated through bidirectional communication between the brain and body, but the mechanisms driving this synchronization are still poorly understood. Astrocytes are a type of glia (non-neuronal) cell in the brain that have been classically described as the brain’s chief homeostatic regulators, but little is known regarding their role in facilitating brain state shifts. Interestingly, astrocyte networks display large, synchronized intracellular calcium events that occur across the entire brain and correlate with pupil dynamics. This suggests that astrocytes may be key players in coordinating the brain with whole-body allostasis, but little research has explored this possibility. Furthermore, the enteric glia surrounding the gut show similarities to astrocytes and could be a conserved mechanism by which whole-body allostasis is achieved. My laboratory aims to bridge astrocyte-linked brain states with allostatic shifts across other organ systems. To do this, we leverage simultaneous in vivo recording of a range of physiological readouts including sympathetic outflow, hormone circulation, cardiovascular dynamics, gut signaling, widefield brain imaging, and behavior in a head-fixed mouse model. We will also model ‘autonomy’ using a quantifiable sensory- motor feedback system to titrate brain states related to environmental control. Over the next five years, I will lead research projects under three main themes: 1) Characterizing brain-body physiology in a stress acclimation model of allostasis; 2) Investigating the role of augmenting environmental control in whole-body allostasis; and 3) Developing new soft-tissue fiber photometry to link enteric glial physiology with whole-body allostasis. Unfortunately, there is no shortage of psychological stress in the world today, especially in medical settings. It is time to bridge our understanding of the brain states underlying psychological stress with whole-body physiology. The allostasis model championed here promises to unify theories and evidence currently isolated to specific organ systems, and will improve individualized medicine, post-operative care, and therapeutic discovery.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Splicing factors (SFs) are RNA-binding proteins that regulate alternative splicing (AS), enabling a single gene to produce a variety of mRNA transcripts and corresponding proteins. AS plays an integral role in development, cancer, and aging, and many SFs are essential for embryonic development. Therefore, SF levels must be tightly controlled to maintain proper gene expression, which can be achieved through the AS of poison exons (PEs) within their own transcripts. PEs within SF transcripts, or SF-PEs, introduce premature termination codons, triggering nonsense-mediated decay (NMD) to reduce SF protein levels, a process known as AS- NMD. Conversely, PE skipping increases SF abundance. Prior studies highlight SF-PEs as critical for cancer cell survival, but their role in non-cancerous cells remains unclear. The goal of this proposal is to determine how SF-PEs maintain SF homeostasis to modulate transcriptomes that sustain pluripotency and differentiation. Our preliminary data suggest that PEs in Srsf3 and Tra2b, two SFs linked to cancer and developmental disease, are essential for pluripotent stem cell survival and embryonic viability. However, the morphological, functional, and transcriptomic effects of PE knockout remain unclear, as does the broader role of SF-PEs in pluripotent stem cell survival. We hypothesize that SF PEs fine-tune pluripotency by buffering SF gene expression and modulating AS of target genes critical for maintaining pluripotent cell viability. Aim 1 will utilize an in vivo reverse genetics approach and long-read RNA sequencing (LR-seq) to characterize how Srsf3- and Tra2b-PEs shape mouse embryonic development. Aim 2 will investigate SF AS-NMD dynamics in vitro using a high-throughput CRISPR-based exon deletion screen to identify SF-PEs essential for iPSC viability. Conditional knockout iPSC models will be engineered to assess effects of SF-PE knockout on transcriptomes using LR-seq, SF target binding using eCLIP, and differentiation phenotypes using functional assays. Successful completion of these Aims will elucidate how SF-PEs modulate transcriptomes, safeguard cell pluripotency, and drive differentiation. This Fellowship will provide me essential training in RNA splicing, stem cell biology, genomics, and scientific communication—critical for my future career as a physician-scientist translating basic research into clinical applications.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Group B Streptococcus (GBS) is commonly associated with neonatal infections and can also cause a variety of soft tissue infections, including urinary tract infections (UTI) in non-pregnant adults. However, patients suffering from type 2 diabetes have a significantly higher incidence of GBS UTIs. Additionally, diabetics are more likely to develop complications from UTIs, such as pyelonephritis, urosepsis, and recurrent UTIs. Due to the metabolic/hormonal imbalance that characterizes diabetes, the disease can result in significant urinary tract alterations, including the presence of glucose and fructose in the urine (glycosuria). As metabolism is a significant regulator of both bacterial virulence and host response, I seek to examine glycosuria as a key contributor to the unique susceptibility of diabetic patients to GBS UTI. To discover how the diabetic urinary environment is shaping GBS adaptation, I conducted an RNA-sequencing (RNA-seq) screen on GBS cultured in urine from diabetic, pre- diabetic, and non-diabetic donors under microoxic conditions. The screen identified numerous genes that connect metabolism to virulence regulation. These results are further supported by my preliminary data showing that glycosuria increases GBS survival against reactive oxygen species and neutrophils. Based on these findings, I hypothesize that diabetic glycosuria modulates GBS virulence and host immune responses to promote urogenital colonization and infection. Testing of this hypothesis will be split into two aims: 1) characterize the fitness and virulence impact of candidate GBS genes in the diabetic urinary environment, and 2) evaluate the role of diabetic-associated urinary carbohydrates on immune response to GBS. I will use diabetic urine and healthy urine supplemented with carbohydrates to delineate the broad effects of diabetes from glycosuria on GBS survival against host factors and host epithelial response using complex in vitro urine-tolerant host models. Additionally, I will employ two mouse models, one of type 2 diabetes and one of glycosuria alone, to assess GBS virulence and the host immune response in vivo. This project will provide me with training in mammalian tissue culture and organoid model systems, mouse models of diabetes and UTI, and approaches to assessing host responses to pathogens while also expanding on my background in microbial pathogenesis. Training will take place at Baylor College of Medicine under the mentorship of experts in GBS, UTIs, and the immune response of bladders to infection. Together, this work will provide critical insights into GBS adaptation in diabetic hosts and inform the development of targeted, non-antibiotic interventions for this high-risk population, with potential implications for other pathogens affecting metabolically dysregulated individuals.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary/Abstract Myelin is a lipid-rich sheath that wraps axons within the vertebrate nervous system to increase the speed of action potential propagation and provide metabolic support to the axon. The myelin sheath is generated by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Developmental myelination is a dynamic and complex process. Some of the cellular interactions driving myelinating glial cell development have been characterized, but a full understanding of the molecular mechanisms that mediate this process is lacking. Genetic disorders affecting the white matter, termed leukodystrophies, result in absent or abnormal myelin development, and are most often diagnosed in infants and children. On the other hand, demyelinating disorders, such as multiple sclerosis, involve the breakdown of existing myelin and can be caused by direct destruction of the oligodendrocyte or occur as a result of neuronal damage or degeneration. All of these disorders can lead to cognitive defects, muscle weakness, motor problems and more. Treatments are limited, and efforts are underway to better understand the mechanisms driving myelin development in order to develop remyelinating therapies. Using publicly available RNA sequencing and proteomics data, the gene cd59 was identified as highly expressed in Schwann cells and oligodendrocytes during development. Cd59 encodes the small GPI-anchored protein Cd59, which is best known for inhibition of complement-induced cell lysis of host cells. Recent studies have also implicated a non-GPI anchored isoform of Cd59 in facilitation of SNARE complex assembly and exocytosis in various cell types. The Kucenas lab generated cd59 mutant zebrafish using CRISPR-Cas9 genome editing and found that Schwann cells of cd59 mutant larvae over-proliferate but display hypomyelination and disrupted node of Ranvier development. This proposal aims to decipher the role of Cd59 in oligodendrocyte development. Aim 1 will further investigate the presence of paranodal bridges linking individual myelin sheaths as seen in preliminary studies and will examine the development and maturation of nodes of Ranvier in cd59 mutants. Aim 2 will focus on understanding the mechanisms by which Cd59 regulates oligodendrocyte development, investigating the role of Cd59 in oligodendrocyte exocytosis and whether this is mediated by a non-GPI anchored isoform as shown in other cell types. This proposal will be conducted in the Kucenas lab at the University of Virginia (UVA). The Kucenas lab has a strong track record of discovering novel mechanisms of glial cell development, and UVA is well known for its expansive glial biology community. Completion of this fellowship will include a mastery of various technical skills, such as molecular biology and advanced microscopy, and drive intellectual and professional growth as an independent scientific researcher.

NIDA - National Institute on Drug Abuse

Project Summary/Abstract Opioid addiction results in the death of tens of thousands of individuals in the U.S. every year. That number is only continuing to grow. A large reason for the growing number of opioid-related deaths is the introduction and mass production of synthetic opioids like fentanyl. Synthetic opioids are much more potent than traditional opioids, and therefore their usage is more often associated with addiction and subsequent death. To combat this growing opioid epidemic, we need a better understanding of the brain regions that propagate opioid and, in particular, synthetic opioid, seeking. One potential critical modulator of opioid seeking is the locus coeruleus (LC). The LC, which is the origin of the primary central noradrenergic (NE) system, is a critical modulator of the somatic symptoms of opioid withdrawal and opioid reinstatement. Additionally, the LC densely express mu opioid receptors (MOR), the target receptor for synthetic opioids. However, despite its dense expression of MOR and its established role in opioid-related behaviors, the LC’s role in opioid seeking is not well established. This proposal aims to establish the role of the LC and, in particular, LC MOR in fentanyl drug seeking. My preliminary data shows that a conditional knockout of MOR on noradrenergic cells potentiates morphine place preference and increases fentanyl consumption in a two-bottle choice paradigm. This data does not, however, implicate a specific brain region in the modulation of these behaviors. The NE system is a vast collection of ascending and descending projections across the brain, and dopamine beta hydroxylase, the rate limiting enzyme in the synthesis of norepinephrine, is also expressed in any epinephrine producing cells, as well as within the sympathetic ganglia, and adrenal glands. We therefore hypothesize that the LC is the region responsible for these findings, and a decrease in LC MOR will also increase fentanyl consumption. In Aim 1 I will determine what chronic fentanyl exposure does to LC MOR; specifically, whether chronic fentanyl exposure downregulates LC MOR. To do so I will implant mice with osmotic minipumps for reliable and consistent delivery of fentanyl over a two-week period. Following this period, brain slices containing the LC will be collected and LC MOR will be assessed both functionally, with the use of whole-cell electrophysiological recordings, and physically, with the use of quantitative polymerase chain reaction. In Aim 2 I will determine whether knockout of LC MOR drives fentanyl consumption. To do so I will first virally knockout LC MOR, after which mice will undergo a two-bottle choice paradigm of fentanyl consumption. I will then test whether our preliminary findings showing that a conditional knockout of NE MOR increases fentanyl consumption are being driven by the LC. Specifically, I will rescue LC MOR expression in these NE MOR conditional knockout mice before they undergo the same two- bottle choice fentanyl consumption paradigm. Overall, this proposal will help establish the role of the LC, and LC MOR specifically, in fentanyl consumption. Furthermore, it will support my growth as an independent scientist ultimately allowing for me to position myself well for a future career in opioid research.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Circumventricular organs (CVOs) are specialized brain regions with unique vascular properties, lacking the classical blood-brain barrier (BBB), to facilitate rapid communication between the brain and periphery. Among these, the median eminence (ME) plays a central role in regulating vital neuroendocrine processes such as hunger, stress, and reproduction. Unlike other CVOs, tanycytes, a specialized ependymoglial cell type, establish a selective barrier between the ME, cerebrospinal fluid, and CNS parenchyma. However, how this barrier is formed, its distinct features compared to other CVOs, and its response to systemic and CNS inflammation remain poorly understood. This project seeks to define the molecular and cellular mechanisms underlying tanycyte barrier formation and its functional role in the ME. Specifically, it will investigate the role of Claudin-10, a key tight junction protein, in maintaining barrier integrity and regulating ion permeability. Additionally, the project will explore how inflammation disrupts tanycyte-mediated barrier function and how such changes affect hypothalamic regulation. These findings will provide critical insights into the dynamics of CNS barriers and their impact on neuroinflammatory and metabolic disorders, offering potential avenues for therapeutic interventions.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Eosinophilic esophagitis (EoE) is a newly described immune-mediated disease and a leading cause of esophageal morbidity in children and young adults. Histologically, pediatric EoE is characterized by esophageal basal cell hyperplasia (BCH) and extensive Th2-associated inflammation. Despite many advances in our understanding of the pathophysiology of EoE, the molecular mechanisms leading to the development of BCH and subsequent epithelial barrier dysfunction in pediatric EoE remain to be elucidated. We previously demonstrated that yes-associated protein 1 (YAP), a key transcriptional regulator in the Hippo signaling pathway, is required for the proliferation and differentiation of basal cells in the developing murine and human esophagus. Using novel pre-weaning mouse models of early-onset EoE and 3D organoid culture systems we have been able to study Th2-driven BCH. Our preliminary data demonstrate nuclear enrichment of YAP in the hyperplastic basal cells of a pre-weaning transgenic mouse model of EoE and esophageal biopsy samples from a pilot study of an EoE patient cohort. In parallel an exploratory untargeted proteomics approach, using human esophageal basal cells (EPC2) cells treated with IL-13 demonstrated that Tenascin-C (TNC), a matrix protein enriched in the basement membrane underlying the hyperplastic basal cells, was among the top-enriched proteins in EPC2 cells upon IL-13 treatment. Moreover, we demonstrated TNC/YAP double-positive cells in our preclinical EoE mouse models as well as increased expression of CD74 in a subpopulation of hyperplastic esophageal basal cells of EoE patients. Based on these findings, the overarching hypothesis is that YAP mediates IL-13-induced CD74 expression, which promotes chronic inflammation characterized by BCH and epithelial barrier dysfunction in pediatric EoE. In Aim 1, we will use YAP loss-of-function mouse models and CD74 inhibitors to test the hypothesis that CD74 activation promotes BCH. In Aim 2, using in vivo and in vitro assays, we will elucidate how CD74 affects the epithelial barrier integrity in the pathogenesis of pediatric EoE. Overall, findings from the proposed studies will significantly advance our understanding of the pathophysiology of pediatric EoE and provide new insights into potential biomarkers and novel therapeutic targets for pediatric EoE.

Islamic Development Bank

Supports science, technology, and innovation projects in IsDB member countries. Funds research, technology transfer, and innovation ecosystem development.

J.W. McConnell Family Foundation

McConnell invests $35M annually in social innovation, reconciliation, and community resilience. Supports systemic change initiatives across Canada.

City of Jacksonville

Jacksonville Water System Improvements

HARRIS CORRECTIONS SOLUTIONS INC

Information Technology Broadcasting and Telecommunications

Friends of Cypher Cafe

The Jefferson Community Center Mock Trial Team will host events that serve as outreach to potential youth participants while providing the community with valuable, accessible information about the judicial system, legal rights, and legal issues that touch our community. Activities will include: producing an educational video; attending competitions; and hosting informative community events and celebration.

City of Richmond

JD SYSTEMS LLC

Jessie Smith Noyes Foundation

Grants for organizations promoting a sustainable and just social and natural environment through food systems and environmental justice.