RESUMO
Social communication guides decision-making, which is essential for survival. Social transmission of food preference (STFP) is an ecologically relevant memory paradigm in which an animal learns a desirable food odour from another animal in a social context, creating a long-term memory1,2. How food-preference memory is acquired, consolidated and stored is unclear. Here we show that the posteromedial nucleus of the cortical amygdala (COApm) serves as a computational centre in long-term STFP memory consolidation by integrating social and sensory olfactory inputs. Blocking synaptic signalling by the COApm-based circuit selectively abolished STFP memory consolidation without impairing memory acquisition, storage or recall. COApm-mediated STFP memory consolidation depends on synaptic inputs from the accessory olfactory bulb and on synaptic outputs to the anterior olfactory nucleus. STFP memory consolidation requires protein synthesis, suggesting a gene-expression mechanism. Deep single-cell and spatially resolved transcriptomics revealed robust but distinct gene-expression signatures induced by STFP memory formation in the COApm that are consistent with synapse restructuring. Our data thus define a neural circuit for the consolidation of a socially communicated long-term memory, thereby mechanistically distinguishing protein-synthesis-dependent memory consolidation from memory acquisition, storage or retrieval.
Assuntos
Tonsila do Cerebelo , Preferências Alimentares , Consolidação da Memória , Memória de Longo Prazo , Comportamento Social , Animais , Masculino , Camundongos , Tonsila do Cerebelo/fisiologia , Tonsila do Cerebelo/citologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Camundongos Endogâmicos C57BL , Odorantes/análise , Bulbo Olfatório/fisiologia , Bulbo Olfatório/citologia , Análise de Célula Única , Sinapses/metabolismo , Transcriptoma , Preferências Alimentares/fisiologia , Preferências Alimentares/psicologiaRESUMO
Animal regeneration involves coordinated responses across cell types throughout the animal body. In endosymbiotic animals, whether and how symbionts react to host injury and how cellular responses are integrated across species remain unexplored. Here, we study the acoel Convolutriloba longifissura, which hosts symbiotic Tetraselmis sp. green algae and can regenerate entire bodies from tissue fragments. We show that animal injury causes a decline in the photosynthetic efficiency of the symbiotic algae, alongside two distinct, sequential waves of transcriptional responses in acoel and algal cells. The initial algal response is characterized by the upregulation of a cohort of photosynthesis-related genes, though photosynthesis is not necessary for regeneration. A conserved animal transcription factor, runt, is induced after injury and required for acoel regeneration. Knockdown of Cl-runt dampens transcriptional responses in both species and further reduces algal photosynthetic efficiency post-injury. Our results suggest that the holobiont functions as an integrated unit of biological organization by coordinating molecular networks across species through the runt-dependent animal regeneration program.
Assuntos
Fotossíntese , Regeneração , Simbiose , Animais , Regeneração/fisiologia , Clorófitas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Early stages of deadly respiratory diseases including COVID-19 are challenging to elucidate in humans. Here, we define cellular tropism and transcriptomic effects of SARS-CoV-2 virus by productively infecting healthy human lung tissue and using scRNA-seq to reconstruct the transcriptional program in "infection pseudotime" for individual lung cell types. SARS-CoV-2 predominantly infected activated interstitial macrophages (IMs), which can accumulate thousands of viral RNA molecules, taking over 60% of the cell transcriptome and forming dense viral RNA bodies while inducing host profibrotic (TGFB1, SPP1) and inflammatory (early interferon response, CCL2/7/8/13, CXCL10, and IL6/10) programs and destroying host cell architecture. Infected alveolar macrophages (AMs) showed none of these extreme responses. Spike-dependent viral entry into AMs used ACE2 and Sialoadhesin/CD169, whereas IM entry used DC-SIGN/CD209. These results identify activated IMs as a prominent site of viral takeover, the focus of inflammation and fibrosis, and suggest targeting CD209 to prevent early pathology in COVID-19 pneumonia. This approach can be generalized to any human lung infection and to evaluate therapeutics.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Macrófagos , Inflamação , RNA Viral , PulmãoRESUMO
Despite gene-expression profiling being one of the most common methods to evaluate molecular dysregulation in tissues, the utilization of cell-free messenger RNA (cf-mRNA) as a blood-based non-invasive biomarker analyte has been limited compared to other RNA classes. Recent advancements in low-input RNA-sequencing and normalization techniques, however, have enabled characterization as well as accurate quantification of cf-mRNAs allowing direct pathological insights. The molecular profile of the cell-free transcriptome in multiple diseases has subsequently been characterized including, prenatal diseases, neurological disorders, liver diseases and cancers suggesting this biological compartment may serve as a disease agnostic platform. With mRNAs packaged in a myriad of extracellular vesicles and particles, these signals may be used to develop clinically actionable, non-invasive disease biomarkers. Here, we summarize the recent scientific developments of extracellular mRNA, biology of extracellular mRNA carriers, clinical utility of cf-mRNA as disease biomarkers, as well as proposed functions in cell and tissue pathophysiology.
Assuntos
Biomarcadores , Ácidos Nucleicos Livres , RNA Mensageiro , Animais , Humanos , Vesículas Extracelulares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory.
Assuntos
Astrócitos , Comunicação Celular , Perfilação da Expressão Gênica , Memória de Longo Prazo , Neurônios , Astrócitos/citologia , Astrócitos/metabolismo , Astrócitos/fisiologia , Complexo Nuclear Basolateral da Amígdala/citologia , Complexo Nuclear Basolateral da Amígdala/metabolismo , Complexo Nuclear Basolateral da Amígdala/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Memória de Longo Prazo/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Análise de Sequência de RNA , Imagem Individual de Molécula , Análise da Expressão Gênica de Célula Única , UbiquitinaçãoRESUMO
Rapid expansion of the pulmonary microvasculature through angiogenesis drives alveolarization, the final stage of lung development that occurs postnatally and dramatically increases lung gas-exchange surface area. Disruption of pulmonary angiogenesis induces long-term structural and physiologic lung abnormalities, including bronchopulmonary dysplasia, a disease characterized by compromised alveolarization. Although endothelial cells are primary determinants of pulmonary angiogenesis, mesenchymal cells (MC) play a critical and dual role in angiogenesis and alveolarization. Therefore, we performed single cell transcriptomics and in-situ imaging of the developing lung to profile mesenchymal cells during alveolarization and in the context of lung injury. Specific mesenchymal cell subtypes were present at birth with increasing diversity during alveolarization even while expressing a distinct transcriptomic profile from more mature correlates. Hyperoxia arrested the transcriptomic progression of the MC, revealed differential cell subtype vulnerability with pericytes and myofibroblasts most affected, altered cell to cell communication, and led to the emergence of Acta1 expressing cells. These insights hold the promise of targeted treatment for neonatal lung disease, which remains a major cause of infant morbidity and mortality across the world.
Assuntos
Displasia Broncopulmonar , Hiperóxia , Células-Tronco Mesenquimais , Recém-Nascido , Lactente , Humanos , Células Endoteliais , PulmãoRESUMO
The assembly and specification of synapses in the brain is incompletely understood1-3. Latrophilin-3 (encoded by Adgrl3, also known as Lphn3)-a postsynaptic adhesion G-protein-coupled receptor-mediates synapse formation in the hippocampus4 but the mechanisms involved remain unclear. Here we show in mice that LPHN3 organizes synapses through a convergent dual-pathway mechanism: activation of Gαs signalling and recruitment of phase-separated postsynaptic protein scaffolds. We found that cell-type-specific alternative splicing of Lphn3 controls the LPHN3 G-protein-coupling mode, resulting in LPHN3 variants that predominantly signal through Gαs or Gα12/13. CRISPR-mediated manipulation of Lphn3 alternative splicing that shifts LPHN3 from a Gαs- to a Gα12/13-coupled mode impaired synaptic connectivity as severely as the overall deletion of Lphn3, suggesting that Gαs signalling by LPHN3 splice variants mediates synapse formation. Notably, Gαs-coupled, but not Gα12/13-coupled, splice variants of LPHN3 also recruit phase-transitioned postsynaptic protein scaffold condensates, such that these condensates are clustered by binding of presynaptic teneurin and FLRT ligands to LPHN3. Moreover, neuronal activity promotes alternative splicing of the synaptogenic Gαs-coupled variant of LPHN3. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways: Gαs signalling and clustered phase separation of postsynaptic protein scaffolds.
Assuntos
Processamento Alternativo , Receptores Acoplados a Proteínas G , Receptores de Peptídeos , Sinapses , Animais , Camundongos , Processamento Alternativo/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Ligantes , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/deficiência , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Sinapses/metabolismo , Transdução de SinaisRESUMO
Preterm birth affects ~10% of pregnancies in the US. Despite familial associations, identifying at-risk genetic loci has been challenging. We built deep learning and graphical models to score mutational effects at base resolution via integrating the pregnant myometrial epigenome and large-scale patient genomes with spontaneous preterm birth (sPTB) from European and African American cohorts. We uncovered previously unidentified sPTB genes that are involved in myometrial muscle relaxation and inflammatory responses and that are regulated by the progesterone receptor near labor onset. We studied genomic variants in these genes in our recruited pregnant women administered progestin prophylaxis. We observed that mutation burden in these genes was predictive of responses to progestin treatment for preterm birth. To advance therapeutic development, we screened ~4000 compounds, identified candidate molecules that affect our identified genes, and experimentally validated their therapeutic effects on regulating labor. Together, our integrative approach revealed the druggable genome in preterm birth and provided a generalizable framework for studying complex diseases.
Assuntos
Nascimento Prematuro , Recém-Nascido , Feminino , Humanos , Gravidez , Nascimento Prematuro/genética , Progestinas , Loci Gênicos , MutaçãoRESUMO
PURPOSE OF REVIEW: This review summarizes the potential of cell-free nucleic acids for predicting preeclampsia, contrasts them with other methods, and discusses these findings' relevance to preeclampsia's pathogenesis and care. RECENT FINDINGS: Recent studies have demonstrated the utility of cell-free nucleic acids in early preeclampsia risk prediction. Encouragingly, nucleic acid measurement exhibits similar or better sensitivity as compared to standard screening assays and furthermore sheds light on preeclampsia's underlying placental biology. Over the past decade, liquid biopsies measuring cell-free nucleic acids have found diverse applications, including in prenatal care. Recent advances have extended their utility to predict preeclampsia, a major cause of maternal mortality. These assays assess methylation patterns in cell-free DNA (cfDNA) or gene levels in cell-free RNA (cfRNA). Currently, preeclampsia care focuses on blood pressure control, seizure prevention, and delivery. If validated, early prediction of preeclampsia through liquid biopsies can improve maternal health and deepen our understanding of its causes.
Assuntos
Ácidos Nucleicos Livres , Hipertensão , Pré-Eclâmpsia , Gravidez , Humanos , Feminino , Ácidos Nucleicos Livres/genética , Placenta , Pressão SanguíneaRESUMO
Hepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk remains unclear. Here, we identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA sequencing uncovered transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development, and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data add a new pathogenesis mechanism-infection of liver stem cells-to the biology of HCV infection that may explain progressive liver damage and enhanced cancer risk through an altered stem cell state.ImportanceThe hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression.
RESUMO
Urine is assayed alongside blood in medicine, yet current clinical diagnostic tests utilize only a small fraction of its total biomolecular repertoire, potentially foregoing high-resolution insights into human health and disease. In this work, we characterized the joint landscapes of transcriptomic and metabolomic signals in human urine. We also compared the urine transcriptome to plasma cell-free RNA, identifying a distinct cell type repertoire and enrichment for metabolic signal. Untargeted metabolomic measurements identified a complementary set of pathways to the transcriptomic analysis. Our findings suggest that urine is a promising biofluid yielding prognostic and detailed insights for hard-to-biopsy tissues with low representation in the blood, offering promise for a new generation of liquid biopsies.
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Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.
Assuntos
Vírus da Dengue , Dengue , Dengue Grave , Criança , Humanos , Linfócitos B , Células Matadoras NaturaisRESUMO
Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.
Assuntos
Alérgenos , Espectrometria de Massas em Tandem , Humanos , Isoformas de Proteínas , Imunoglobulina E/metabolismo , Antígenos de Plantas/metabolismoRESUMO
Despite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic laboratories. Manual sample preparation remains a slow, expensive and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA using one of two commercially available, inexpensive and open-source robotic systems: the Opentrons OT1.0 or OT2.0. Like many RNA isolation protocols, ours can be decomposed into three subparts: RNA extraction, DNA digestion and RNA cleaning and concentration. RT-qPCR data using a synthetic spike-in confirms comparable RNA quality to the gold standard, manual sample processing. The semi-automated pipeline also shows improvement in sample throughput (+12×), time spent (-11×), cost (-3×) and biohazardous waste produced (-4×) compared with its manual counterpart. This protocol enables cell-free RNA extraction from 96 samples simultaneously in 4.5 h; in practice, this dramatically improves the time to results, as we recently demonstrated. Importantly, any laboratory already has most of the parts required (manual pipette and corresponding tips and kits for RNA isolation, cleaning and concentration) to build a semi-automated sample processing pipeline of their own and would only need to purchase or three-dimensionally print a few extra parts (US$5.5 K-12 K in total). This pipeline is also generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research laboratories.
Assuntos
Ácidos Nucleicos Livres , DNA , RNA , Manejo de Espécimes/métodosRESUMO
The impact of next-generation sequencing (NGS) cannot be overestimated. The technology has transformed the field of life science, contributing to a dramatic expansion in our understanding of human health and disease and our understanding of biology and ecology. The vast majority of the major NGS systems today are based on the concept of 'sequencing by synthesis' (SBS) with sequential detection of nucleotide incorporation using an engineered DNA polymerase. Based on this strategy, various alternative platforms have been developed, including the use of either native nucleotides or reversible terminators and different strategies for the attachment of DNA to a solid support. In this review, some of the key concepts leading to this remarkable development are discussed.
Assuntos
DNA , Nucleotídeos , Humanos , Análise de Sequência de DNA , DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Aging is characterized by a decline in tissue function, but the underlying changes at cellular resolution across the organism remain unclear. Here, we present the Aging Fly Cell Atlas, a single-nucleus transcriptomic map of the whole aging Drosophila. We characterized 163 distinct cell types and performed an in-depth analysis of changes in tissue cell composition, gene expression, and cell identities. We further developed aging clock models to predict fly age and show that ribosomal gene expression is a conserved predictive factor for age. Combining all aging features, we find distinctive cell type-specific aging patterns. This atlas provides a valuable resource for studying fundamental principles of aging in complex organisms.
Assuntos
Envelhecimento , Senescência Celular , Drosophila melanogaster , Animais , Envelhecimento/genética , Perfilação da Expressão Gênica , Transcriptoma , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Atlas como AssuntoRESUMO
BACKGROUND: Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multifocal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear, and therapeutic options are limited. METHODS: We performed cell-free messenger RNA (cf-mRNA) sequencing to characterize the circulating transcriptome of PSC and noninvasively investigate potentially bioactive signals that are associated with PSC. Serum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with NAFLD. Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were developed using PSC dysregulated cf-mRNA genes. RESULTS: Differential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and healthy controls or NAFLD shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, HSCs, and KCs, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster, which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA diagnostic classifier using liver-specific genes that discriminated PSC from healthy control subjects using gene transcripts of liver origin. CONCLUSIONS: Blood-based whole-transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in sera of subjects with PSC, which may be used to diagnose patients with PSC. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for noninvasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.
Assuntos
Ácidos Nucleicos Livres , Colangite Esclerosante , Colestase , Hepatopatia Gordurosa não Alcoólica , Humanos , Secretoma , RNA MensageiroRESUMO
Prenatal screening using sequencing of circulating cell-free DNA has transformed obstetric care over the past decade and significantly reduced the number of invasive diagnostic procedures like amniocentesis for genetic disorders. Nonetheless, emergency care remains the only option for complications like preeclampsia and preterm birth, two of the most prevalent obstetrical syndromes. Advances in noninvasive prenatal testing expand the scope of precision medicine in obstetric care. In this review, we discuss advances, challenges, and possibilities toward the goal of providing proactive, personalized prenatal care. The highlighted advances focus mainly on cell-free nucleic acids; however, we also review research that uses signals from metabolomics, proteomics, intact cells, and the microbiome. We discuss ethical challenges in providing care. Finally, we look to future possibilities, including redefining disease taxonomy and moving from biomarker correlation to biological causation.
Assuntos
Ácidos Nucleicos Livres , Teste Pré-Natal não Invasivo , Nascimento Prematuro , Recém-Nascido , Gravidez , Feminino , Humanos , Testes Genéticos/métodos , Aneuploidia , Ácidos Nucleicos Livres/genética , RNA , Nascimento Prematuro/genéticaRESUMO
At birth, the lung is still immature, heightening susceptibility to injury but enhancing regenerative capacity. Angiogenesis drives postnatal lung development. Therefore, we profiled the transcriptional ontogeny and sensitivity to injury of pulmonary endothelial cells (EC) during early postnatal life. Although subtype speciation was evident at birth, immature lung EC exhibited transcriptomes distinct from mature counterparts, which progressed dynamically over time. Gradual, temporal changes in aerocyte capillary EC (CAP2) contrasted with more marked alterations in general capillary EC (CAP1) phenotype, including distinct CAP1 present only in the early alveolar lung expressing Peg3, a paternally imprinted transcription factor. Hyperoxia, an injury that impairs angiogenesis induced both common and unique endothelial gene signatures, dysregulated capillary EC crosstalk, and suppressed CAP1 proliferation while stimulating venous EC proliferation. These data highlight the diversity, transcriptomic evolution, and pleiotropic responses to injury of immature lung EC, possessing broad implications for lung development and injury across the lifespan.