RESUMO
Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks, and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious and time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelization. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 min of hands-on time and 4.5 h of machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the plug and play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We show high-quality ChIP-seq on hundreds to a few thousand of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naive and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the two-cell stage of embryonic development. PnP-ChIP-seq allows nonexpert laboratories worldwide to conveniently run robust, standardized ChIP-seq, whereas its high throughput, consistency, and sensitivity pave the way toward large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.
Assuntos
Histonas , Microfluídica , Animais , Cromatina , Imunoprecipitação da Cromatina/métodos , Sequenciamento de Cromatina por Imunoprecipitação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/genética , Camundongos , Reprodutibilidade dos TestesRESUMO
Aquaculture is a critical food source for the world's growing population, producing 52% of the aquatic animal products consumed. Marine aquaculture (mariculture) generates 37.5% of this production and 97% of the world's seaweed harvest. Mariculture products may offer a climate-friendly, high-protein food source, because they often have lower greenhouse gas (GHG) emission footprints than do the equivalent products farmed on land. However, sustainable intensification of low-emissions mariculture is key to maintaining a low GHG footprint as production scales up to meet future demand. We examine the major GHG sources and carbon sinks associated with fed finfish, macroalgae and bivalve mariculture, and the factors influencing variability across sectors. We highlight knowledge gaps and provide recommendations for GHG emissions reductions and carbon storage, including accounting for interactions between mariculture operations and surrounding marine ecosystems. By linking the provision of maricultured products to GHG abatement opportunities, we can advance climate-friendly practices that generate sustainable environmental, social, and economic outcomes.
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Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.
Assuntos
Proteínas Cromossômicas não Histona/análise , Ensaios de Triagem em Larga Escala/economia , Histonas/análise , Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/análise , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Polarização de Fluorescência , Histonas/química , Histonas/metabolismo , Humanos , Ligantes , Técnicas Analíticas Microfluídicas/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
We argue that the recommendations made by the Institute of Medicine's 2011 report, Chimpanzees in Biomedical and Behavioral Research: Assessing the Necessity, are methodologically and ethically confused. We argue that a proper understanding of evolution and complexity theory in terms of the science and ethics of using chimpanzees in biomedical research would have had led the committee to recommend not merely limiting but eliminating the use of chimpanzees in biomedical research. Specifically, we argue that a proper understanding of the difference between the gross level of examination of species and examinations on finer levels can shed light on important methodological and ethical inconsistencies leading to ignorance of potentially unethical practices and policies regarding the use of animals in scientific research.
Assuntos
Experimentação Animal/ética , Evolução Biológica , Pesquisa Biomédica/métodos , Modelos Animais , National Academies of Science, Engineering, and Medicine, U.S., Health and Medicine Division , Pan troglodytes , Animais , Pesquisa Biomédica/ética , Compreensão , Ética em Pesquisa , Relatório de Pesquisa , Estados UnidosRESUMO
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
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.
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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.
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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
Food systems and the communities they support are increasingly challenged by climate change and the need to arrest escalating threats through mitigation and adaptation. To ensure climate change mitigation strategies can be implemented effectively and to support substantial gains in greenhouse gas emissions reduction, it is, therefore, valuable to understand where climate-smart strategies might be used for best effect. We assessed mariculture in 171 coastal countries for vulnerabilities to climate change (12 indicators) and opportunities to deliver climate mitigation outcomes (nine indicators). We identified Northern America and Europe as having comparatively lower regional vulnerability and higher opportunity for impact on climate mitigation. Australia, Canada, France, Italy, Japan, Republic of Korea, New Zealand, Norway and the United States of America were identified as well-positioned to advance strategies linked to mariculture. However, the nature of vulnerabilities and opportunities within and between all regions and countries varied, due to the formation of existing mariculture, human development factors and governance capacity. Our analysis demonstrates that global discussion will be valuable to motivating climate-smart approaches associated with mariculture, but to ensure these solutions contribute to a resilient future, for industry, ecosystems and communities, local adaptation will be needed to address constraints and to leverage local prospects. This article is part of the theme issue 'Nurturing resilient marine ecosystems'.
Assuntos
Ecossistema , Gases de Efeito Estufa , Aclimatação , Adaptação Fisiológica , Mudança Climática , HumanosRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease observed with aging that represents the most common form of dementia. To date, therapies targeting end-stage disease plaques, tangles, or inflammation have limited efficacy. Therefore, we set out to identify a potential earlier targetable phenotype. Utilizing a mouse model of AD and human fetal cells harboring mutant amyloid precursor protein, we show cell intrinsic neural precursor cell (NPC) dysfunction precedes widespread inflammation and amyloid plaque pathology, making it the earliest defect in the evolution of the disease. We demonstrate that reversing impaired NPC self-renewal via genetic reduction of USP16, a histone modifier and critical physiological antagonist of the Polycomb Repressor Complex 1, can prevent downstream cognitive defects and decrease astrogliosis in vivo. Reduction of USP16 led to decreased expression of senescence gene Cdkn2a and mitigated aberrant regulation of the Bone Morphogenetic Signaling (BMP) pathway, a previously unknown function of USP16. Thus, we reveal USP16 as a novel target in an AD model that can both ameliorate the NPC defect and rescue memory and learning through its regulation of both Cdkn2a and BMP signaling.
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Doença de Alzheimer , Doenças Neurodegenerativas , Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Senescência Celular , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Transgênicos , Placa Amiloide , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
In diabetes, glucagon secretion from pancreatic α cells is dysregulated. The underlying mechanisms, and whether dysfunction occurs uniformly among cells, remain unclear. We examined α cells from human donors and mice using electrophysiological, transcriptomic, and computational approaches. Rising glucose suppresses α cell exocytosis by reducing P/Q-type Ca2+ channel activity, and this is disrupted in type 2 diabetes (T2D). Upon high-fat feeding of mice, α cells shift toward a "ß cell-like" electrophysiological profile in concert with indications of impaired identity. In human α cells we identified links between cell membrane properties and cell surface signaling receptors, mitochondrial respiratory chain complex assembly, and cell maturation. Cell-type classification using machine learning of electrophysiology data demonstrated a heterogenous loss of "electrophysiologic identity" in α cells from donors with type 2 diabetes. Indeed, a subset of α cells with impaired exocytosis is defined by an enrichment in progenitor and lineage markers and upregulation of an immature transcriptomic phenotype, suggesting important links between α cell maturation state and dysfunction.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagon , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Tipo 2/metabolismo , Exocitose/fisiologia , Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , CamundongosRESUMO
Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.
Assuntos
Atlas como Assunto , Células , Especificidade de Órgãos , Splicing de RNA , Análise de Célula Única , Transcriptoma , Linfócitos B/metabolismo , Células/metabolismo , Humanos , Especificidade de Órgãos/genética , Linfócitos T/metabolismoRESUMO
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
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Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Transcriptoma , Animais , Núcleo Celular/metabolismo , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes de Insetos , Masculino , RNA-Seq , Caracteres Sexuais , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.
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Variações do Número de Cópias de DNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Cromossomos Humanos X/genética , Dosagem de Genes , Humanos , Receptores Acoplados a Proteínas G/genética , Reprodutibilidade dos TestesRESUMO
Most cell fate trajectories during development follow a diverging, tree-like branching pattern, but the opposite can occur when distinct progenitors contribute to the same cell type. During this convergent differentiation, it is unknown if cells 'remember' their origins transcriptionally or whether this influences cell behavior. Most coronary blood vessels of the heart develop from two different progenitor sources-the endocardium (Endo) and sinus venosus (SV)-but whether transcriptional or functional differences related to origin are retained is unknown. We addressed this by combining lineage tracing with single-cell RNA sequencing (scRNAseq) in embryonic and adult mouse hearts. Shortly after coronary development begins, capillary endothelial cells (ECs) transcriptionally segregated into two states that retained progenitor-specific gene expression. Later in development, when the coronary vasculature is well established but still remodeling, capillary ECs again segregated into two populations, but transcriptional differences were primarily related to tissue localization rather than lineage. Specifically, ECs in the heart septum expressed genes indicative of increased local hypoxia and decreased blood flow. Adult capillary ECs were more homogeneous with respect to both lineage and location. In agreement, SV- and Endo-derived ECs in adult hearts displayed similar responses to injury. Finally, scRNAseq of developing human coronary vessels indicated that the human heart followed similar principles. Thus, over the course of development, transcriptional heterogeneity in coronary ECs is first influenced by lineage, then by location, until heterogeneity declines in the homeostatic adult heart. These results highlight the plasticity of ECs during development, and the validity of the mouse as a model for human coronary development.
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Vasos Coronários/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células Endoteliais/metabolismo , Animais , Humanos , Camundongos , RNA-Seq , Análise de Célula ÚnicaRESUMO
Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.
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Drosophila melanogaster/metabolismo , Neuritos/metabolismo , Nervo Olfatório/metabolismo , Transcriptoma , Animais , Análise de Célula Única , Fatores de TempoRESUMO
Recognition of environmental cues is essential for the survival of all organisms. Transcriptional changes occur to enable the generation and function of the neural circuits underlying sensory perception. To gain insight into these changes, we generated single-cell transcriptomes of Drosophila olfactory- (ORNs), thermo-, and hygro-sensory neurons at an early developmental and adult stage using single-cell and single-nucleus RNA sequencing. We discovered that ORNs maintain expression of the same olfactory receptors across development. Using receptor expression and computational approaches, we matched transcriptomic clusters corresponding to anatomically and physiologically defined neuron types across multiple developmental stages. We found that cell-type-specific transcriptomes partly reflected axon trajectory choices in development and sensory modality in adults. We uncovered stage-specific genes that could regulate the wiring and sensory responses of distinct ORN types. Collectively, our data reveal transcriptomic features of sensory neuron biology and provide a resource for future studies of their development and physiology.
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Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Neurônios Receptores Olfatórios/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Feminino , Masculino , Análise de Sequência de RNA , Análise de Célula Única , Olfato , TranscriptomaRESUMO
BACKGROUND: The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable. METHODS: We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array. RESULTS: The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors. CONCLUSIONS: The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.
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DNA de Neoplasias/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Análise Mutacional de DNA , Progressão da Doença , Receptores ErbB/antagonistas & inibidores , Feminino , Genoma Humano/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Células Tumorais CultivadasRESUMO
Copy number variations (CNVs) in the human genome are conventionally detected using high-throughput scanning technologies, such as comparative genomic hybridization and high-density single nucleotide polymorphism (SNP) microarrays, or relatively low-throughput techniques, such as quantitative polymerase chain reaction (PCR). All these approaches are limited in resolution and can at best distinguish a twofold (or 50%) difference in copy number. We have developed a new technology to study copy numbers using a platform known as the digital array, a nanofluidic biochip capable of accurately quantitating genes of interest in DNA samples. We have evaluated the digital array's performance using a model system, to show that this technology is exquisitely sensitive, capable of differentiating as little as a 15% difference in gene copy number (or between 6 and 7 copies of a target gene). We have also analyzed commercial DNA samples for their CYP2D6 copy numbers and confirmed that our results were consistent with those obtained independently using conventional techniques. In a screening experiment with breast cancer and normal DNA samples, the ERBB2 gene was found to be amplified in about 35% of breast cancer samples. The use of the digital array enables accurate measurement of gene copy numbers and is of significant value in CNV studies.