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  • Epigenetic Enablers of Meningioma Growth

    Berry, Bethany C (2024-04-26)
    Meningiomas are the most common primary intracranial brain tumor, often causing significant disability and sometimes even death. The most aggressive meningiomas commonly exhibit extensive genomic disruption that can lead to genotoxic and proteotoxic stress, but the mechanisms that enable these tumors to thrive are unknown. To date, meningiomas have no effective chemotherapy. This study used a high-throughput bioactive small molecule screen of established meningioma cell lines and RNA-sequencing of patient meningiomas to identify EHMT2/G9a inhibitors as potent cytotoxic agents in meningioma in vitro. Further, studies using the small molecule EHMT2/G9a inhibitor, UNC0631 demonstrated reduced tumor growth in an orthotopic xenograft mouse model of meningioma in vivo. We used CUT&Tag and transcriptomic analyses of established meningioma cell lines after EHMT2/G9a inhibition to identify the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress apoptotic signaling pathway as key factors in EHMT2/G9a-mediated meningioma cell death. After EHMT2/G9a inhibition, we observed a collapse of heat shock protein expression and hypothesized that downregulation of a deacetylase, Sirtuin 1 (SirT1), may be responsible. Consistent with decreased SirT1 activity, Heat Shock Factor 1 exhibited increased lysine acetylation and decreased DNA binding at the promoters of downregulated heat shock proteins. Overexpression of SirT1 or shRNA-mediated knockdown of the ER stress response mediators, ATF4 or CHOP/DDIT3, decreased meningioma cell death caused by EHMT2/G9a inhibition. The molecular chaperone and ER stress inhibitor, 4-phenylbutyric acid, abrogated meningioma cell death occurring after EHMT2/G9a inhibition. In conclusion, epigenetic maintenance of heat shock protein activity and suppression of the UPR/ER stress apoptotic signaling pathway by EHMT2/G9a and SirT1 are essential for aggressive meningioma growth.
  • Regulation of Lipolysis by 14-3-3 Proteins on Human Adipocyte Lipid Droplets

    Yang, Qin (2024-04-11)
    Lipid droplets (LDs) in adipocytes are pivotal for systemic lipid metabolism, serving as storage centers during nutritional surplus and as sources of fatty acids when energy is needed. These LDs react to hormonal stimuli like catecholamines and insulin, and their impaired response can lead to dysregulated lipolysis, lipotoxicity, and an increased risk of metabolic diseases. The specific mechanisms behind lipid release in human adipocytes remain largely unexplored. This study aims to elucidate the control of lipid mobilization in human adipocytes. We utilized advanced techniques to generate and differentiate primary progenitor cells on a large scale. Employing proximity labeling with enhanced ascorbate peroxidase (APEX2), we identified the interactome of perilipin 1 (PLIN1), a key LD component protein, under various lipolytic states. Through LC-MS/MS, we discovered 70 proteins interacting specifically with PLIN1. This includes PNPLA2 and LIPE, vital for regulated triglyceride hydrolysis, and four 14-3-3 protein family members (YWHAB, YWHAE, YWHAZ, YWHAG), which are known to regulate diverse signaling pathways. Our functional studies revealed that YWHAB is essential for maximal cyclic adenosine monophosphate (cAMP)-stimulated lipolysis, as its CRISPR-Cas9-mediated knockout mitigates lipolysis through a mechanism independent of insulin signaling. In summary, our use of proximity labeling not only comprehensively mapped the LD proteome in human adipocytes but also unveiled new regulatory mechanisms in adipocyte lipolysis control, specifically involving 14-3-3 proteins.
  • A Pilot Clinical Trial of an Informatics-Informed Mobile Transitional Care Program: the Paramedic Assisted Community Evaluation after Discharge (PACED) Intervention

    O'Connor, Laurel (2024-04-03)
    Introduction: Early rehospitalization of frail older adults after hospital discharge is detrimental to patients and hospital systems. Implementing effective strategies to execute a feasible and effective transitional care plan is challenging. Mobile integrated health (MIH) programs, which deploy mobile assets into the community to care for patients, may present a possible solution to facilitating effective transitional care back to home environments after hospitalization. However, there have been few previous studies investigating MIH models for transitional care delivery. The objective of this project is to assess the implementation and effectiveness of an informatics-supported paramedic-led MIH transitional care program for frail older adults. Methods: Patients 65 and older preparing for discharge from the hospital with an eFrailty index of 0.24 or greater were enrolled to participate in a structured post-discharge transitional home visit conducted by community paramedics within 72 hours of discharge. Demographic and clinical information, as well as healthcare utilization patterns, were recorded at enrollment and 30 days after the index hospitalization. Additionally, a separate control group of patients that were screened for the intervention but excluded due to geographical location of residence were aggregated and their electronic health record data including demographical and outcomes data was abstracted. Categorical group comparisons were conducted using chi-square tests and continuous variables group comparisons were conducted using the Kruskal–Wallis equality-of-populations rank test. Crude and adjusted binomial regressions were used for comparative outcomes. Results: In total 100 subjects were enrolled in the intervention (median age 81, 64% female) and 47 were included in the control group (median age 80, 55.2% female). The recruitment rate was 18.0%. The complete intervention protocol was completed and documented by paramedics for 90 (90.0%) patients. The crude and relative risk of 30-day rehospitalization was decreased in the PACED group compared to the control (RR=0.40, CI 0.19-0.84, p=0.03). There was a non-significant trend toward decreased risk of 30-day ED visits (RR=0.61, CI=0.37-1.37, p=0.23). Paramedics identified medication errors in 34 (34.0%) of the participants; the errors were remediated during the visit in 31 (91.2%). Additionally, 67 (67.0%) of subjects screened positive for high fall risk and 7 subjects (7.0%) screened positive for delirium. Conclusions: This pilot study of MIH intervention transition care program programs was feasible with high protocol fidelity and yields preliminary evidence that the intervention results in a decreased risk of rehospitalization in frail older adults.
  • Bacterial Patterns of Pathogenesis in Intestinal Immunity

    Tse-Kang, Samantha Y (2024-04-03)
    Mechanisms of pathogen recognition in barrier tissues, which interface with both commensal and virulent bacteria, are critical for health and not fully understood. Here, I define two mechanisms by which the nematode Caenorhabditis elegans identifies an infectious pathogen to activate innate immunity. First, we characterized a central role for lysosome-related organelles in innate immune sensing within intestinal tissues. We found that lysosome-related organelles are actively maintained to limit p38 MAPK activity. We discovered that TIR-1/SARM1, a conserved immune signaling regulator, localized to the membranes of lysosome-related organelles. Upon exposure to a redox active virulence effector produced by Pseudomonas aeruginosa, pyocyanin (PYO), these organelles collapsed and alkalinized in a manner that induced TIR-1/SARM1 aggregation and p38 MAPK activation. Second, we identified the first bacterial pattern recognition receptor in C. elegans. We found that C. elegans detected another toxic phenazine, phenazine-1-carboxamide (PCN), to activate the transcription of innate immune genes. We identified that PCN was a ligand for the nuclear hormone receptor, NHR-86. Binding of PCN to NHR-86 activated an anti-pathogen program in the intestine. In addition, the production of PCN correlated to the virulence of P. aeruginosa strains. Together, these data identify phenazines as bacterial patterns of pathogenesis that are sensed by nematodes to recognize the presence of toxigenic bacteria. C. elegans animals mount distinct innate immune pathways against specific phenazine metabolites to ensure a coordinated and robust response against infectious agents. The findings herein define new mechanisms of pathogen recognition that inform the evolution of innate immune pathways in metazoans.
  • Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

    Min, Kyounghee; Yenilmez, Batuhan; Kelly, Mark; Echeverria, Dimas; Elleby, Michael; Lifshitz, Lawrence M; Raymond, Naideline; Tsagkaraki, Emmanouela; Harney, Shauna M; DiMarzio, Chloe; et al. (2024-04-02)
    Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.
  • Investigation of the Structural Mechanisms of a Bacterial Clamp Loader

    Landeck, Jacob T (2024-04-01)
    The sliding clamp is an integral protein in DNA replication and repair, where it increases the speed of DNA synthesis and serves as a scaffold for repair proteins. Since the sliding clamp is a closed ring, clamp loaders must open the ring and then close it around DNA. The clamp loader and sliding clamp are the only two components of the replisome that are conserved across all three domains of life. However, differences in their structures and biochemical activities suggest that the mechanisms of opening and loading differ between prokaryotes and eukaryotes. Structures of the eukaryotic clamp loader clearly illustrate its clamp loading mechanism, but there were no comparable structures for bacterial clamp loaders. To understand how mechanisms of bacterial clamp loaders compare to their eukaryotic counterparts, I determined a series of structures of the E. coli clamp loader at distinct stages in clamp loading. To understand how ATP binding enables the clamp loader to bind to the sliding clamp, I determined a structure of the E. coli clamp loader bound to a non-hydrolyzable ATP analog. I found that the E. coli clamp loader opens its sliding clamp at a single pivot point into a planar conformation, but transitions to a helical conformation upon binding primer-template (p/t)-junctions. This behavior contrasts with eukaryotic clamp loaders, which open their sliding clamp through multiple pivot points into a helical conformation before binding p/t-junctions. My work also revealed that like the eukaryotic clamp loader, the E. coli clamp loader does not need to undergo a conformational change to close the sliding clamp on p/t-junctions. Furthermore, I explored how the E. coli clamp loader is inhibited by the bacteriophage protein gene product 8. This dissertation explores the structural mechanisms used by the bacterial clamp loader and illuminates similarities and differences between clamp loaders across the domains of life.
  • Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements

    Wirthlin, Morgan E; Schmid, Tobias A; Elie, Julie E; Zhang, Xiaomeng; Kowalczyk, Amanda; Redlich, Ruby; Shvareva, Varvara A; Rakuljic, Ashley; Ji, Maria B; Bhat, Ninad S; et al. (2024-03-29)
    Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.
  • Deep learning as a tool to better understand transcription factor binding across cell types and species

    Andrews, Gregory (2024-03-29)
    Deep learning has transformed our everyday lives. Facebook facial recognition, Netflix personalized recommendations and ChatGPT are all powered by deep learning. Neural networks, inspired by human intelligence, are the workhorse of deep learning, and are capable of learning complex relationships and patterns within large amounts of heterogeneous data. However, they are often referred to as black boxes due to the complex nature of these patterns and the difficulty involved in discerning them. This work seeks to open this metaphorical black box and leverage the information learned by these networks to better understand transcription factor (TF) binding. Convolutional neural networks (CNNs) excel at learning patterns from images. Nucleotide sequences are simply 1D images, a fact we use to develop a CNN-based motif discovery algorithm that outperforms classical and other deep learning-based approaches. We use our method and thousands of publicly available TF ChIP-seq experiments to annotate the binding sites of 367 human TFs. We then investigated the evolutionary conservation of these TF binding sites (TFBSs) in the mammalian lineage. We next demonstrate CNNs can be used to predict TF binding across cell types using only sequence and chromatin accessibility data. Lastly, we highlight the application of CNNs in unsupervised learning, specifically in the context of clustering brain specific regulatory elements based on sequence features. Altogether, the results presented herein highlight the importance of carefully constructing and training CNNs to achieve state of the art performance and gain the most biologically meaningful insights when trained on regulatory genomic data.
  • Sex differences in time to initiate NSAIDs or bDMARDs among patients with axial spondyloarthritis

    Shridharmurthy, Divya; Lapane, Kate L; Baek, Jonggyu; Nunes, Anthony P; Weisman, Michael H; Kay, Jonathan; Liu, Shao-Hsien (2024-03-27)
    Objective: We evaluated sex differences in time to initiation of nonsteroidal anti-inflammatory agents (NSAIDs) or biologic disease modifying antirheumatic drugs (bDMARDs) among patients with axial spondyloarthritis (axSpA). Methods: Using the 2013-2018 IBM® MarketScan® Database, we identified 174,632 axSpA patients aged ≥18 years. We evaluated the time between axSpA diagnosis and the first prescription NSAID dispensing (among those with no baseline NSAIDs use) or bDMARDs infusion/procedure claim (among those dispensed ≥ two different prescription NSAIDs in the baseline period). Adjusted hazard ratios (aHR) for time to initiation of NSAIDs or bDMARDs were computed using survival analyses. Cox proportional hazard models estimated associations between sex and predictors of treatment initiation. Results: Average age at diagnosis was 48.2 years, 65.7% were females, and 37.8% had ≥ one NSAIDs dispensing before axSpA diagnosis. Of those without dispensing for ≥ two different prescription NSAIDs before diagnosis, NSAIDs were initiated earlier in females than males (NSAID initiators: Females (32.9%), Males (29.3%); aHR: 1.14, 95% CI: 1.11-1.16). Among those with ≥ two different prescription NSAIDs dispensations in the baseline period, 4.2% initiated a bDMARD while 77.9% continued NSAIDs use after diagnosis. Time to bDMARDs initiation was longer for females than males (aHR:0.61, 95% CI:0.52-0.72), but bDMARDs were initiated sooner among those with NSAIDs use in the baseline period. Conclusion: Prescription NSAID use was more common than initiation of bDMARDs among patients newly diagnosed with axSpA. Females appeared more likely to continue NSAIDs after diagnosis, and the time to initiation of bDMARDs was longer for females than males.
  • Implementation of Medications for Opioid Use Disorder in Jail Settings and the Post-release Risk Environment

    Michener, Pryce S. (2024-03-26)
    Background: People with opioid use disorder (OUD) who have experienced incarceration are at disproportionate risk of opioid-related mortality, particularly in the first two weeks post-release. Methods: We analyzed factors associated with implementation of medications for OUD (MOUD) in jails and the post-release environment. The specific aims were to evaluate: 1) the association between random and observed urine drug screening (UDS) practices and long-term retention in opioid treatment providers (OTPs); 2) the association between diffusion of MOUD in jails and organizational factors; and 3) perceptions of post-release overdose risk among people who received MOUD while incarcerated. Results: OTPs that utilized random and observed UDS on ≥90% of clients had 13% lower long-term retention rates compared to OTPs which did not highly utilize these practices. Perspectives from people with lived experience of carceral MOUD treatment revealed a high degree of knowledge of risks. Exposure to the toxic drug supply, underdosing of MOUD, and lack of social support were associated with increased overdose risk. In jails, high organizational change capacity was associated with a 2- to 3-fold increase in the likelihood that jail staff would have positive perceptions and high knowledge of MOUD. A high degree of education for jail staff was associated with higher likelihood of positive perceptions and high knowledge of MOUD.
  • Mechanistic Role of the Calcium Channel TRPC6 in Driving Aggressive Traits in Triple-Negative Breast Cancer

    Mukhopadhyay, Dimpi (2024-03-26)
    Despite advances in the diagnosis and therapy of breast cancer, issues continue to drive morbidity and mortality, especially tumor cells that persist after chemotherapy and contribute to recurrence and metastasis. The presence of such persister cells is particularly relevant for triple negative breast cancer (TNBC) because it harbors a relatively high frequency of cells with properties of cancer stem cells (CSCs) that resist standard-of-care therapies. My interest is understanding the nature of persister cells in TNBC and identifying novel mechanisms that contribute to persistence and stemness that can be exploited to improve therapy. In my first project, I identified a calcium channel TRPC6 that is enriched specifically in CSCs, including cells that are quiescent and persist after chemotherapy, and that has a causal role in promoting resistance. The mechanism by which it functions in this capacity involves its ability to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated Ca2+ entry represses the epithelial splicing factor epithelial splicing regulatory protein 1 (ESRP1), which enables expression of the integrin α6B splice variant. This integrin splice variant has been implicated in sustaining breast CSCs. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic targeting of TRPC6 using a specific chemical inhibitor sensitizes TNBC cells, organoids, and patient-derived xenografts (PDX) to chemotherapy by impeding the splicing of α6 integrin mRNA and inducing Myc. These data revealed a Ca2+-dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing. In my second project, I extended my investigation of TRPC6 to cell metabolism based on my observation that TRPC6 inhibition sensitized TNBC cell lines and organoids to ferroptosis, a form of cell death that involves iron-dependent lipid peroxidation of cell membranes. This observation piqued my interest because there is evidence that the ability to resist ferroptosis facilitates metastasis. In pursuit of the mechanism by which TRPC6 promotes ferroptosis resistance, I discovered that it maintains a level of reduced glutathione (GSH) that is sufficient to buffer oxidative stress. More specifically, I observed that the TRPC6-driven quiescent population has a low biosynthetic demand that enables GSH levels to be maintained by intracellular cysteine biosynthesis without relying on extracellular uptake through the xCT amino acid transporter. Based on these data, I propose that TRPC6 facilitates metastasis by enabling metastatic cells to resist ferroptosis. In summary, this thesis details how a specific calcium channel (TRPC6) contributes to the function of persister/CSCs in TNBC to promote therapy resistance and metastasis, and that it can be targeted in vivo to improve the therapy of TNBC.
  • Enhancing Safety and Efficacy of Genome Editing In Vivo with Compact Cas9 and Guide Chemical Modification

    Zhang, Han (2024-03-25)
    Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) have revolutionized science and medicine. However, applying the CRISPR/Cas system for in vivo therapeutic genome editing remains challenging due in part to delivery obstacles. Adeno-associated virus (AAV) vectors remain one of the most promising vehicles for gene therapies, particularly in extrahepatic tissues. Nevertheless, employing AAV for in vivo CRISPR/Cas genome editing presents difficulties, including limited packaging capacity, prolonged expression, redosing complexities, modest multiplexing capability, dose-related toxicity, and immunogenicity. This thesis addresses AAV limitations in delivering CRISPR/Cas in vivo in two ways. First, I focus on developing a novel delivery modality in which the CRISPR RNA (crRNA) is delivered in the form of a naked oligonucleotide, separate from AAV-expressed effector and trans-activating crRNA (tracrRNA). I find that a short, fully stabilized oligonucleotide (a ‘protecting oligo’) can significantly enhance the potency, stability, and uptake of a heavily chemically modified crRNA. The establishment of AAV/crRNA co-delivery modality offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation. Second, I use a compact Nme2Cas9 adenine base editor (Nme2-ABE) to engineer a single AAV delivery system for base editing in vivo. The capability to deliver a base editor via a single AAV holds the potential to enhance safety by minimizing immune responses, mitigating dose-related toxicity, and reducing manufacturing complexity.
  • Modular vector assembly enables rapid assessment of emerging CRISPR technologies

    McGee, Abby V; Liu, Yanjing V; Griffith, Audrey L; Szegletes, Zsofia M; Wen, Bronte; Kraus, Carolyn; Miller, Nathan W; Steger, Ryan J; Escude Velasco, Berta; Bosch, Justin A; et al. (2024-03-13)
    The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
  • Elucidating the Role of Ciliary Proteins INPP5E and IFT43 in Photoreceptor Function and Retinal Disease

    Gupta, Mohona (2024-03-11)
    Like primary cilia, photoreceptor outer segments require a constant flux of intracellular transport of proteins and lipids for their renewal since they lack synthetic machinery. A disruption in this transport can result in retinal degradation and vision loss. This research aims to delineate how genes implicated in retinal ciliopathies support photoreceptor vitality. Intraflagellar Transport Protein (IFT's) role in material transport through the connecting cilium is pivotal for outer segment health. While the significance of IFT88, an IFT-B complex protein is recognized for outer segment development (Pazour et al., 2002a), the functions of IFT-A components in photoreceptor physiology have not been fully understood. Human populations with retinal degeneration contain mutations in IFT43, a subunit of the IFT-A complex. After its deletion in rod photoreceptors in mice, we observed rapid photoreceptor degeneration along with huge amount, whereas C11orf74, an IFTAP (Intraflagellar Transport Associated Protein), didn't significantly damage photoreceptors when absent. It is evident from these findings that IFT43 plays a key role in maintaining the structural and functional integrity of photoreceptors. Mutations in INPP5E cause a wide range of retinopathies, from syndromic conditions such as Joubert's syndrome to isolated blindness. Systemic deletion of INPP5E in mice is lethal, leading to pronounced developmental abnormalities in photoreceptors. Conditional Inpp5e knockout mice displayed disruption of outer segment formation, diminished disk assembly, compromised actin architecture, disrupted Golgi, and increased extracellular vesicle release. These pathologies, along with aberrant rhodopsin distribution illustrate INPP5E's essential role in photoreceptor segment maintenance and cargo transport regulation.
  • Elucidating TFEB’s Role in Transcriptional Regulation of Macrophage Phenotype and Response

    Honwad, Havisha H. (2024-03-08)
    Diverse inputs dictate macrophage transcriptional programming that impacts activation and relevant function, under homeostatic and disease conditions. My work addresses how stress-responsive transcription factor EB (TFEB) regulates macrophage gene expression following bacterial infection or exposure to cholinergic drugs. Using TFEB deficient macrophages, we show that TFEB is critical for mounting pro-inflammatory responses to Staphylococcus aureus infection. Mechanistically, the NADPH oxidase (PHOX)-dependent oxidative burst triggers TFEB activation, involving CD38 and NAADP-mediated Ca2+ release from intracellular stores. These findings reveal a previously unknown pathway, linking bacterial phagocytosis to macrophage phenotype and function via TFEB. In the absence of infection, we show that treatment of naïve macrophages with cholinergic agent PNU282987, an α7 nicotinic acetylcholine receptor (α7nACHR) agonist, induces pro-inflammatory gene expression, increasing Tnf, Ifnb1, Il1b and Il6 mRNA levels, without translating to increased cytokine secretion. However, primary macrophages and RAW264.7 cells showed no detectable α7nAChR expression, challenging the proposed α7nACHR -specific mode of action. We propose that PNU282987 may function through a non-receptor mediated pathway by activating stress responsive factor TFEB that involves ROS/H2O2 generation, implicating the MCOLN1-calcineurin pathway. These data offer critical insights into the pharmacological effects of PNU282987 beyond conventional receptor-based mechanisms. Overall, we describe novel mechanistic insights for macrophage TFEB activation as an integrator of distinct inputs, with important implications for inflammation and homeostasis.
  • Introducing Telomere-Dysfunction Induced Cellular Senescence in iPSC-Based Modeling Of Neurodegenerative Diseases

    Neherin, Kashfia (2024-03-06)
    Investigation of disease-associated cellular changes in vitro has leaped forward significantly with the innovation of induced pluripotent stem cell (iPSC) technology. However, resetting the epigenetic landscape and aging clock during reprogramming results in iPSC-differentiated cells resembling fetal cell types instead of adult or aged cells. The lack of cellular aging in iPSC-based models presents a significant drawback in the investigation of age-associated diseases such as Alzheimer’s disease. My thesis aims to introduce proper cellular aging to improve iPSC-based modeling of neurodegeneration. Toward this goal, I created an inducible system to trigger senescence in iPSC-based cell models, as recent studies showed senescence playing a crucial role in aging and neurodegeneration. I utilized CRISPR-interference (CRISPRi) to suppress telomere repeat factor 2 (TERF2), a significant component of the telomere-protecting Shelterin complex. I demonstrated that suppression of TERF2 in iPSCs robustly activated DNA damage response (DDR), p53/p21 signaling, and cellular senescence in an inducible and synchronized manner. The inducible approach allows temporal control of senescence activation throughout differentiation from iPSCs to desired cell types. I applied the CRISPRi-TERF2 approach to iPSC-differentiated neural progenitor cells (NPCs) and showed that suppression of TERF2 efficiently activated DDR, p53/p21 signaling, and cellular senescence in differentiated NPCs. This inducible model of cellular senescence generated in this study will enable the investigation of cellular senescence using isogenic comparisons in the progression of age-associated neurodegeneration and improve disease modeling with a proper cellular aging context to facilitate drug discovery.
  • Social engagement and cognitive impairment among nursing home residents: The role of sensory impairment

    Xu, Shu; Jesdale, William M; Dubé, Catherine E; Nielsen, Natalia N; McPhillips, Emily A; Lapane, Kate L (2024-03-05)
    Background and objectives: Using US national nursing home data, this cross-sectional study sought to evaluate 1) the association between lack of social engagement and level of cognitive impairment; and 2) the extent to which this association differs by hearing and visual impairment. Research design and methods: Our sample included 793,846 nursing home residents aged ≥ 50 years. The Index of Social Engagement was categorized as none/lower (0, 1, 2) or higher levels (3 through 6). Cognitive Performance Scale was grouped as intact/mild (0, 1, 2), moderate (3, 4), or severe (5, 6). Multinomial models provided adjusted odds ratio (aOR) and 95 % confidence intervals (CI) between none/lower social engagement and cognitive impairment. We estimated relative excess risk due to interaction (RERI) to quantify the joint effects of social engagement and sensory impairment types. Results: Overall, 12.6 % had lower social engagement, 30.3 % had hearing impairment, and 40.3 % had visual impairment. Compared to residents with high social engagement, those with lower social engagement were more likely to have moderate/severe cognitive impairment (aORmoderate = 2.21, 95 % CI 2.17-2.26; aORsevere = 6.49, 95 % CI 6.24-6.74). The impact of low social engagement on cognitive impairment was more profound among residents with hearing impairment and/or visual impairment (RERIhearing = 3.89, 95 % CI 3.62-4.17; RERIvisual = 25.2, 95 % CI 23.9-26.6)). Discussion and implications: Residents with lower social engagement had higher levels of cognitive impairment. Residents with sensory impairments are potentially more susceptible to the negative impact of lower levels of social engagement on level of cognitive impairment.
  • Targeting Intracellular Mycobacterium Tuberculosis by Exploiting Host-Pathogen Interactions

    Jaecklein, Eleni Elizabeth (2024-03-01)
    Infinite combinations of complex interactions between host, pathogen, and environment generate considerable heterogeneity in the disease states of those infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). This high degree of disease heterogeneity makes TB particularly challenging to treat and necessitates lengthy treatment regimens. A better understanding of the factors that impact antibiotic activity (i.e. pharmacodynamics) and exposure (i.e pharmacokinetics) is needed to improve TB treatment. Here we investigated the effect of host immunity on mycobacterial respiration to provide a better understanding of how host pressures can affect the performance of antimycobacterial drugs. We show that cytochrome bd oxidase in Mtb is required for fitness in acidic environments like those found in the phagosome of IFNγ-activated macrophages. These acidic environments alter the respiratory requirements of Mtb consequently affecting the efficacy of respiratory inhibitors under these conditions, demonstrating a potential synergy between host immunity and drug PD. Separately, we conducted a proof-of-concept study using glucan lipid particles (GLPs) as a generalizable, inhalable drug delivery platform for antimycobacterial compounds. We aimed to alter the PK of existing TB antibiotics by creating formulations that better localize drugs to the site of infection, slow-release, and limit systemic drug exposure. GLPs can accommodate a variety of payloads, can be aerosolized, and are selectively taken up by macrophages. These qualities make them an attractive TB drug delivery platform. Using novel packaging techniques, we stably and efficiently loaded both first- and second-line TB therapeutics into GLPs. We show that intranasal delivery of GLP-encapsulated clofazimine reduced Mtb burden in vivo and increased drug retention in the lung. Together these studies highlight the multifactorial nature of drug performance in TB. Each of these studies focuses on the impact of the host on drug PK or PD whether by targeting drug delivery to immune cells or identifying critical interactions between immunity and bacterial physiology, respectively. This work provides insights and tools for future development of TB therapies.
  • Silencing Parkinson's risk allele Rit2 sex-specifically compromises motor function and dopamine neuron viability

    Kearney, Patrick J; Zhang, Yuanxi; Liang, Marianna; Tan, Yanglan; Kahuno, Elizabeth; Conklin, Tucker L; Fagan, Rita R; Pavchinskiy, Rebecca G; Shaffer, Scott A; Yue, Zhenyu; et al. (2024-02-23)
    Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself impacts DA neuron function and/or viability. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide clear evidence that Rit2 loss is causal for SNc cell death and motor dysfunction, and reveal key sex-specific differences in the response to Rit2 loss.
  • Racial and Ethnic Disparities in Use of Colorectal Cancer Screening Among Adults With Chronic Medical Conditions: BRFSS 2012-2020

    Castañeda-Avila, Maira A; Tisminetzky, Mayra; Oyinbo, Atinuke G; Lapane, Kate L (2024-02-22)
    Introduction: People with chronic conditions and people with colorectal cancer (CRC) may share common risk factors; thus, CRC screening is important for people with chronic conditions. We examined racial and ethnic differences in the use of CRC screening among people with various numbers of chronic conditions. Methods: We included data on adult respondents aged 50 to 75 years from the Behavioral Risk Factor Surveillance System in 2012 through 2020. We categorized counts of 9 conditions as 0, 1, 2, 3, and ≥4. We classified self-reported CRC screening status as up to date or not. We used Poisson models to estimate adjusted prevalence ratios (APRs) among the different counts of chronic conditions in 4 racial and ethnic groups: Hispanic adults with limited English proficiency (LEP), Hispanic adults without LEP, non-Hispanic Black adults, and non-Hispanic White adults. Results: Overall, 66.5% of respondents were up to date with CRC screening. The prevalence of being up to date increased with the number of chronic conditions. We found disparities among racial and ethnic groups. Hispanic respondents with LEP had lower rates than non-Hispanic White adults of being up to date with CRC screening across all counts of chronic conditions (APR for 0 conditions = 0.67; 95% CI, 0.64-0.71; APR for ≥4 conditions = 0.85; 95% CI, 0.79-0.91). Hispanic respondents without LEP with 0, 1, or 2 conditions were less likely than non-Hispanic White respondents to be up to date with CRC screening. We found no significant differences between non-Hispanic Black and non-Hispanic White respondents. Conclusion: We found disparities among Hispanic BRFSS respondents with LEP, who had lower rates than non-Hispanic White respondents of being up to date with CRC screening, regardless of the number of chronic conditions. Tailored interventions are needed to address these disparities and improve screening rates, particularly among Hispanic people.

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