Now showing items 1-20 of 5268

    • Kinetics of Pre-mRNA 3’ End Cleavage

      Torres Ulloa, Leslie (2024-05-31)
      3’ end cleavage and polyadenylation are required steps in pre-mRNA maturation. The rate at which 3’ end cleavage occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. While there are numerous high-throughput methods for global profiling of RNA maturation rates, the study of pre-mRNA 3’ end cleavage kinetics has remained limited to low-throughput approaches, and the temporal regulation of polyadenylation site choice that determines the composition of the 3’ UTRs of mRNAs remains poorly understood. This research project seeks to address this gap by introducing a novel genome-wide, site-specific methodology for estimating rates of pre-mRNA 3’ end cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in-silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. In Drosophila melanogaster S2 cells, we find that cleavage rates are fast but highly variable across sites, with alternative events being slowest. This variability in rates is underpinned by distinctive sequence elements, where an A-rich region upstream of the cleavage site, a U-rich element downstream of the cleavage site, and a higher density of polyadenylation signals, lead to faster cleavage reactions. Assessment of Polymerase II dynamics around cleavage sites reveals that cleavage rates are associated with the localization of RNA Polymerase II at the end of a gene and faster cleavage leads to quicker degradation of downstream read-through RNA. This approach for estimating pre-mRNA 3’ end cleavage kinetics opens new possibilities in the study of co-transcriptional regulation of mRNA expression and transcription termination across cellular states.
    • Variation in Depth of Sedation Targeted and Achieved among Mechanically Ventilated Patients and Associated Outcomes

      Rucci, Justin M (2024-05-30)
      Introduction: Sedative agents are commonly administered to patients receiving mechanical ventilation (MV). Practice guidelines recommend provision of light sedation within validated scoring systems (e.g., Richmond Agitation Sedation Scale [RASS]), but recognize some circumstances require deeper sedation. The real-world approaches to depth of sedation, and the impact of hospital sedation practices on patient outcomes, remain uncharacterized. Methods: We used the US based eICU collaborative research database to identify adult patients who received MV > 24 hours, who did not have a diagnosis that may require sedatives for indications other than facilitating MV, and who had recorded RASS goals and scores. We used mixed effects regression models to determine factors associated with initial RASS goals and rates of RASS score-goal concordance. We organized hospitals into quartiles of risk-adjusted RASS score-goal concordance, and used g-computation to evaluate differences in ventilator free days (VFD) at hospital day 28. Results: We identified a study sample of 1,650 adult patients (at 21 hospitals) who met inclusion/exclusion criteria. Hospital-level risk-adjusted initial RASS goals ranged from -1.4 to 0.2, and hospital-level risk-adjusted RASS score-goal concordance ranged from 27% to 64%. Patients admitted to hospitals in the highest quartile of score-goal concordance (quartile 4) were generally targeted for deeper sedation (median RASS goal -1.31) than patients admitted to hospitals in the lowest quartile (quartile 1) (median RASS goal -0.58). Compared to patients admitted to quartile 1 hospitals, patients at quartile 4 hospitals experienced fewer VFDs (adjusted incidence risk difference -2.4, 95% CI -4.26 to -0.36). Conclusion: US hospitals prescribe RASS goals in line with guideline recommendations for light sedation, but there is wide variation in achieving these RASS goals. Hospitals with higher RASS score-goal concordance typically prescribed deeper RASS goals, and patients admitted to these hospitals experienced fewer VFD.
    • Spatial Transcriptomics Reconstruction of Mouse Olfactory System

      Wang, I-Hao (2024-05-20)
      The olfactory system is crucial for animals in tasks such as foraging, mate selection, and predator avoidance due to its ability to detect and distinguish a vast array of environmental chemicals. Mice detect these chemicals via olfactory receptor (OR) proteins, which are uniquely expressed by olfactory sensory neurons (OSNs); each OSN expresses only one OR type. OSNs with the same OR converge their axons to a specific location in the olfactory bulb (OB), forming a structure known as a glomerulus. This precise organization ensures a consistent, spatially invariant pattern of glomerular activation for each odorant, playing a likely role in the brain's decoding of odor identities. Nevertheless, the exact locations of most glomeruli are unknown, and the mechanisms that create consistent glomerular maps across different animals are not fully understood. In this study, we leveraged spatial transcriptomics and machine learning to map the majority of glomerular positions within the mouse OB. Furthermore, single-cell RNA sequencing revealed distinct transcriptional profiles for each OSN type, characterized not only by their OR gene but also by a unique set of axon guidance genes. These profiles can predict the eventual location of each OSN's glomerulus within the olfactory bulb. We also identified a correlation between the spatial distribution of glomeruli and the characteristics of their corresponding ORs, suggesting a chemotopic arrangement in the mouse olfactory system. Additionally, we probed the complexity of the OB by creating a spatially resolved cell atlas through spatial single-cell transcriptomics, revealing the identity and distribution of neuron subtypes that contribute to odor perception.
    • Exploring the relationship between school-supervised asthma therapy and social determinants of health in pediatric asthma care

      Al-Halbouni, Layana; Ryan, Grace W; Radu, Sonia; Spano, Michelle; Sabnani, Reshma; Phipatanakul, Wanda; Gerald, Lynn B; Garg, Arvin; Pbert, Lori; Trivedi, Michelle (2024-05-16)
      Background: Social determinants of health (SDoH), including access to care, economic stability, neighborhood factors, and social context, strongly influence pediatric asthma outcomes. School-supervised asthma therapy (SST) is an evidence-based strategy that improves asthma outcomes, particularly for historically marginalized children, by providing support for daily medication adherence in school. However, little is known about the relationship between these programs and the adverse SDoH commonly affecting underrepresented minority and marginalized children with asthma. Methods: We examined qualitative data from interviews (n = 52) conducted between 2017 and 2020 with diverse multi-level partners involved in Asthma Link, a SST intervention. Participants included end-users (children and their parents), deliverers (school nurses and pediatric providers), and systems-level partners (e.g., insurers, legislators, and state officials). We used inductive coding to determine themes and subthemes and deductive coding using the Healthy People 2030 SDoH framework. Results: Three themes emerged: (1) SST mitigates adverse SDoH (improves access to preventive healthcare and asthma health literacy), (2) SST benefits children experiencing specific adverse SDoH (provides a consistent medication routine to children with unstable family/housing situations) and (3) specific adverse SDoH impede SST implementation (economic instability, culture and language barriers). Conclusion: This study suggests an important relationship between SDoH and SST that warrants further evaluation in our future work on this community-based asthma intervention. Moreover, our findings underscore the importance of measuring SDoH in the implementation and evaluation of pediatric asthma interventions, particularly given the strong influence of these social factors on child health outcomes.
    • Identifying Therapeutic Oligonucleotide-Induced Neurotoxicity and Methods for Safe Delivery to the Central Nervous System

      Miller, Rachael (2024-04-29)
      Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by an autosomal dominant mutation in Exon 1 of the Huntingtin gene (Htt). There are no approved treatments for HD. Oligonucleotide therapeutics (ASOs and siRNAs) offer a new strategy to treat genetically defined CNS diseases. These therapeutics aim to attenuate disease pathogenesis by targeting Htt mRNA to reduce the toxic mutant protein. Recent technological advancements now enable robust distribution and efficacy throughout mouse, sheep, and NHP brains. However, oligonucleotides can cause acute neurotoxicity when injected directly into the CSF. This dissertation aims to optimize oligonucleotide delivery for the treatment of HD by addressing safety issues across species. We used electroencephalography (EEG) and electromyography (EMG) in awake animals to confirm that direct CSF injection of oligonucleotides induces seizures. We hypothesized that this was due to the negatively charged oligonucleotides changing the delicate balance of divalent cations in the CSF. To address this issue, we developed an artificial CSF (aCSF) buffer supplemented with Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+ in the injected solution to prevent the imbalance. Real-time EEG monitoring in awake mice and lambs confirmed the absence of seizures when oligonucleotides were delivered in the new aCSF buffer. In summary, this dissertation identified a potential cause of oligonucleotide-induced acute neurotoxicity, developed a method to safely deliver oligonucleotides to the CNS with Ca2+/Mg2+-enriched buffers, and demonstrated the viability of this formulation in a large animal model. These findings support a new method for safely delivering oligonucleotides to the CNS to treat neurological diseases.
    • 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.
    • Advancing Oligonucleotide Technologies for Malignant Brain Tumors and Other Central Nervous System Diseases

      Sarli, Samantha L (2024-04-26)
      Oligonucleotides are a class of synthetic, nucleic acid-based drugs that modulate the expression of disease-causing genes. These drugs are chemically modified to ensure safe and effective activity in relevant tissues in vivo. However, in complex tissues such as the brain, oligonucleotides can show striking differences in activity across cell types. Determining the activity profile of an oligonucleotide in distinct cell types can inform on mechanism of action and is key in moving these drugs towards the clinic. In this thesis, I broadly focused on developing tools to measure efficacy and improve the safety of gene-silencing oligonucleotides in the central nervous system (CNS). Much of this work was dedicated to glioblastoma multiforme (GBM), a universally lethal brain tumor that is largely resistant to current surgical and drug interventions. To this end, I designed a method to measure gene silencing by oligonucleotides in GBM xenografts versus normal brain tissue in vivo. I assessed the impact of conjugates on oligonucleotide activity patterns in tumor and normal brain cells and identified conjugates with potential GBM applications. In addition, I studied formulation as a strategy to mitigate acute neurotoxicity induced by antisense oligonucleotides (ASOs) delivery to the CNS. I also optimized immunostaining methods for a phosphorothioate-specific antibody to characterize biodistribution of monovalent and multivalent ASOs. In summary, this thesis expands the frameworks used in the rational design of these drugs and further
    • Deciphering Splicing Anomalies in Pancreatic Islets and Cancer Cells

      MacMillan, Hannah J (2024-04-25)
      Efficient splicing hinges upon an intricate balance between splice site choice accuracy and temporal coordination. It is well known that splicing programs are widely dysregulated in disease contexts such as diabetes and cancer, but the mechanism and consequence is not always clear. Here, we investigate splicing outcomes in the context of SNPs and splicing factor mutations. Specifically, we examine back-splicing of the antisense non-coding RNA in the INK locus (ANRIL) in cardiometabolic disease and then query the consequences of recurrent splicing factor mutations SF3B1K700E and U2AF1S34F in cancer. We characterize consistent circular ANRIL isoforms across dozens of pancreatic islet donors and find that individuals with a Type II Diabetes T2D risk-SNP in exon 2 of ANRIL produce more circANRIL. We also find that a higher circular:linear ANRIL isoform ratio is associated with decreased beta cell proliferation, drawing an association between back-splicing and disease phenotype. Outside of transcriptomic variation due to individual SNPs relating to cardiometabolic disease, we investigate the consequences of missense mutations within core splicing factors in cancer. Through a combination of nascent and steady state RNA sequencing and mathematical modeling, we find that both highly prevalent mutations in MDS and leukemias, SF3B1K700E and U2AF1S34F, cause a significant global alteration in splicing rate. Our result suggests that prevalent spliceosomal mutation not only contributes to disease progression in the form of select mis-spliced transcripts as previously reported, but also through a global disruption in the temporal coordination of mRNA processing. Together, our research sheds further light on the interplay between aberrant splicing mechanisms and the progression of disease phenotypes.
    • Mechanisms of Interferon-α/β Receptor (IFNAR) Dependent and Independent Autoimmune Diabetes in LEW.1WR1 Rats

      Arowosegbe, Adediwura (2024-04-22)
      Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing pancreatic β cells by immune cells, leading to insulin deficiency. T1D is driven by intricate interactions between the innate and adaptive immune mechanism, and autoreactive T-cells perpetuate destruction of islets cells following priming by proinflammatory cytokines and chemokines. Although the primary risk factor for T1D is genetic, environmental factors have been implicated as possible triggers or accelerators in the pathogenesis of T1D. Viral infections, especially enteroviruses have been implicated in the pathogenesis of T1D. Mechanisms proposed for such association include virus-induced innate immune responses including type I interferon (IFN) that unmask β-cells for recognition by autoreactive T-cells. Type I IFN has been implicated in the early stages of T1D autoimmunity and previous studies in our rat model highlight the essential role of virus-induced, type I IFN responses as rats lacking type I IFN signaling (Ifnar1-/- LEW.1WR1 rats) have delayed onset and up to 50% reduction in the incidence of autoimmune diabetes. The goal of my thesis research is to delineate type I IFN dependent and independent mechanisms that drive autoimmune diabetes using LEW.1WR1 rats, models in which autoimmune diabetes can be induced with combined poly I:C and virus infection. I hypothesize that in Ifnar1-/- LEW.1WR1 rats, type II IFN and non-interferon innate immune responses compensate for type I IFN responses and drive the adaptive immune cells to mediate autoimmune diabetes. Transcriptome profiles of wild type (WT) and Ifnar1-/- LEW.1WR1 rat islets over a time course were analyzed to define temporal transcriptional events that lead to autoimmune diabetes in prediabetic LEW.1WR1 rats following poly I:C and KRV treatment. Pancreatic sections of treated rats were also analyzed using RNA-in situ hybridization (RNA-ISH) to spatially map islet cells inflammation and to correlate with local islet T cell recruitment. In WT LEW.1WR1 rats, a transcriptional signature characterized by interferon-stimulated genes, chemokines, major histocompatibility class I, and genes for the ubiquitin-proteasome system was identified in subsets of β and α cells. These signature cells increased in frequency over time and correlated with local islet T cell recruitment. Type I IFN genes as well as genes for the ubiquitin-proteasome system are largely suppressed in poly I:C + KRV treated Ifnar1-/- rats compared to control Ifnar1-/- rats, while MHC class I genes were upregulated in poly I:C + KRV treated Ifnar1-/- rats compared to control Ifnar1-/- rats. Cytokine analysis reveal an increase in levels of IFNγ in poly I:C + KRV treated Ifnar1-/- rats compared to WT rats. Blocking IL-1R does not protect from diabetes in WT and Ifnar1-/- treated rats although both are protected with depletion of CD8+ T cells. Infiltration of immune cells in WT rats was accompanied with extensive islet damage while infiltration in Ifnar1-/- rats was less destructive. Inhibition of the JAK signaling pathway confirmed the requirement of the JAK-STAT signaling pathway and CD8+ T cells in progression to diabetes in the WT rats and further testing to confirm this requirement in Ifnar1-/- rats is ongoing. Although CCL proteins were expressed on immune cells, blockade of CCR5 had no effect on diabetes incidence. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, innate immune responses in the early phase of autoimmune diabetes pathogenesis.
    • CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators

      Jiang, Hao-Ching; Park, Sung Jin; Wang, I-Hao; Bear, Daniel M; Nowlan, Alexandra; Greer, Paul L (2024-04-18)
      The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.
    • Quantitative Metabolic Analysis of Brown Adipose Tissue, Skeletal Muscle, and Liver During Cold and Diet Induced Thermogenesis

      Haley, John A (2024-04-12)
      Non-shivering thermogenesis by brown adipose tissue (BAT) is an adaptive mechanism for maintaining body temperature in cold environments, critical in rodents and human infants, and has substantial influence on adult human metabolism. Stimulating BAT therapeutically is also being investigated as a strategy against metabolic diseases because of its ability to function as a catabolic sink, through the thermogenic protein, uncoupling protein 1 (UCP1). Thus, understanding how BAT uses nutrients to fuel its demanding metabolism has both basic and translational implications. Here we developed an arteriovenous (AV) metabolomics technique which we used to quantify metabolite exchange between BAT and skeletal muscle during cold exposure in fed male mice, identifying unexpected metabolite utilization between organs. Of note, glucose and lactate provide ~85% of carbon during chronic cold exposure and that cold and CL316,243 initiate divergent fuel utilization profiles. We identified a novel role for glutamine synthesis during thermogenesis to avoid ammonia buildup and promote fuel oxidation. We then set out to investigate a lesser studied thermogenic process termed diet-induced thermogenesis (DIT), which is initiated through prolonged consumption of high-fat diets (HFD). Here, we found that UCP1KO male mice develop liver fibrosis after HFD feeding but female mice do not. We also found that DIT transcriptionally diverges from cold-induced thermogenesis, however, DIT metabolically prefers glucose as a major fuel source accounting for ~53% of carbon consumption. Together these data present a comprehensive landscape of BAT fuel utilization during cold- and diet-induced thermogenesis providing a framework to guide further translational studies.
    • 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.
    • Distinct Members of the C. elegans CeMbio Reference Microbiota Exert Cryptic Virulence that is Masked by Host Defense

      Gonzalez, Xavier (2024-04-08)
      Microbiotas are complex microbial communities that colonize specific niches in the host and provide essential organismal functions that are important in health and disease. Understanding the ability of each community member to promote or impair host health, alone or in the context of the community, is imperative for understanding how differences in community structure affect host health. Here, we turn to the simple organism C. elegans, a powerful whole- animal model, to study host-microbiota interactions. We show the differential ability of individual C. elegans microbiota members to activate innate immunity through the conserved PMK-1/p38 MAPK, ACh-WNT, and HLH-30/TFEB pathways. Although distinct microbiota members differed in their ability to activate the conserved pathways, the ability to activate did not correlate with decreased lifespan in wild type or immunodeficient animals. These results suggest that the microbiota of C. elegans is rife with bacteria that can shorten the host’s lifespan if host defense is compromised. The nervous system innervates barrier tissues and regulates the immune response. How neurons detect microbes and subsequently regulate host immune response remains an active area of investigation. We show that cholinergic signaling can regulate HLH-30/TFEB activation. In addition, we find S. aureus infection decreases ASH-mediated avoidance behavior and causes ASH neuronal degeneration. These data support the C. elegans nervous system as a critical microbial sensor and regulator of host defense. These findings can be translated to advance ongoing research of complex intestinal-microbiota and neuronal-microbe interactions in humans, and how individual microbiota members promote or impair host health.
    • Association of perinatal depression and postpartum contraception intent, choice, and actual use

      Masters, Grace A; Julce, Clevanne; Carroll, Smita; Person, Sharina D.; Allison, Jeroan J.; Byatt, Nancy; Moore Simas, Tiffany A (2024-04-05)
      Objectives: Depression is common during pregnancy and the year following childbirth (the perinatal period). This study assessed the association of depressive symptoms and contraception decisions in perinatal individuals. Study design: We conducted a secondary analysis using data from the PRogram in Support of Moms (PRISM) study, a cluster randomized controlled trial of active interventions which aimed to address perinatal depression. This analysis included 191 individuals aged 18-45 who screened positive for depression on the Edinburgh Postnatal Depression Scale (EPDS, score ≥10) during pregnancy or up to 3 months postpartum. We assessed contraception intent and method choice at 1-3 months postpartum. At 5-7 months postpartum, we assessed contraceptive method used and EPDS depression scores. We used logistic regressions to examine the relationship between depression and contraceptive use/method. Results: At 1-3 months postpartum, the majority of participants (76.4%) expressed an intention to use contraception. Of those, over half (53.4%) indicated a preference for higher effectiveness contraception methods. Participants with persistent depression symptoms (positive EPDS) at 5-7 months were significantly less likely to report using higher effectiveness contraceptive methods (aOR = 0.28, 95% CI = 0.11-0.70) compared to those without. Among participants with persistent depressive symptoms, 21.1% reported using a contraception method of lower effectiveness than had originally intended. Conclusion: Perinatal individuals with persistent depressive symptoms at 5-7 months postpartum reported greater use of less-effective contraception methods than originally planned. Implications: We found associations between perinatal depression and use of less effective contraception use. Provider discussions regarding contraception planning is important, particularly in those with perinatal depression symptoms.
    • 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.
    • Dopamine control of social novelty preference is constrained by an interpeduncular-tegmentum circuit

      Molas, Susanna; Freels, Timothy G; Zhao-Shea, Rubing; Lee, Timothy; Gimenez-Gomez, Pablo; Barbini, Melanie; Martin, Gilles E; Tapper, Andrew R (2024-04-03)
      Animals are inherently motivated to explore social novelty cues over familiar ones, resulting in a novelty preference (NP), although the behavioral and circuit bases underlying NP are unclear. Combining calcium and neurotransmitter sensors with fiber photometry and optogenetics in mice, we find that mesolimbic dopamine (DA) neurotransmission is strongly and predominantly activated by social novelty controlling bout length of interaction during NP, a response significantly reduced by familiarity. In contrast, interpeduncular nucleus (IPN) GABAergic neurons that project to the lateral dorsal tegmentum (LDTg) were inhibited by social novelty but activated during terminations with familiar social stimuli. Inhibition of this pathway during NP increased interaction and bout length with familiar social stimuli, while activation reduced interaction and bout length with novel social stimuli via decreasing DA neurotransmission. These data indicate interest towards novel social stimuli is encoded by mesolimbic DA which is dynamically regulated by an IPN→LDTg circuit to control NP.
    • Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer

      Mishra, Alok K; Ye, Tianyi; Banday, Shahid; Thakare, Ritesh P; Su, Chinh Tran-To; Pham, Ngoc N H; Ali, Amjad; Kulshreshtha, Ankur; Chowdhury, Shreya Roy; Simone, Tessa M; et al. (2024-04-03)
      CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.
    • Combinatorial expression of neurexin genes regulates glomerular targeting by olfactory sensory neurons [preprint]

      Park, Sung Jin; Wang, I-Hao; Lee, Namgyu; Jiang, Hao-Ching; Uemura, Takeshi; Futai, Kensuke; Kim, Dohoon; Macosko, Evan; Greer, Paul (2024-04-02)
      Precise connectivity between specific neurons is essential for the formation of the complex neural circuitry necessary for executing intricate motor behaviors and higher cognitive functions. While trans -interactions between synaptic membrane proteins have emerged as crucial elements in orchestrating the assembly of these neural circuits, the synaptic surface proteins involved in neuronal wiring remain largely unknown. Here, using unbiased single-cell transcriptomic and mouse genetic approaches, we uncover that the neurexin family of genes enables olfactory sensory neuron (OSNs) axons to form appropriate synaptic connections with their mitral and tufted (M/T) cell synaptic partners, within the mammalian olfactory system. Neurexin isoforms are differentially expressed within distinct populations of OSNs, resulting in unique pattern of neurexin expression that is specific to each OSN type, and synergistically cooperate to regulate axonal innervation, guiding OSN axons to their designated glomeruli. This process is facilitated through the interactions of neurexins with their postsynaptic partners, including neuroligins, which have distinct expression patterns in M/T cells. Our findings suggest a novel mechanism underpinning the precise assembly of olfactory neural circuits, driven by the trans -interaction between neurexins and their ligands.
    • 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.